Lung cancer markers and uses thereof

ABSTRACT

Methods and compositions are provided for assessing (e.g., diagnosing), treating, and preventing diseases, especially cancer, and particular lung cancer, using lung cancer markers (LCM). Individual LCM and panels comprising multiple LCM are provided for these and other uses. Methods and compositions are also provided for determining or predicting the effectiveness of a treatment or for selecting a treatment using LCM. Methods and compositions are further provided for modulating cell function using LCM. Also provided are compositions that modulate LCM (e.g., antagonists or agonists), such as antibodies, proteins, small molecule compounds, and nucleic acid agents (e.g., RNAi and antisense agents), as well as pharmaceutical compositions thereof. Further provided are methods of screening for agents that modulate LCM, and agents identified by these screening methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/346,547, filed on Nov. 8, 2016 (and issued as U.S. Pat. No.10,338,075 on Jul. 2, 2019), which is a continuation of U.S. applicationSer. No. 14/331,803, filed on Jul. 15, 2014 which is a divisional ofU.S. application Ser. No. 13/005,031, filed on Jan. 12, 2011 (and issuedas U.S. Pat. No. 8,808,997 on Aug. 19, 2014), which is a divisional ofU.S. application Ser. No. 12/273,994, filed on Nov. 19, 2008 (and issuedas U.S. Pat. No. 7,892,760 on Feb. 22, 2011), which claims the benefitof U.S. provisional application Ser. No. 61/003,767, filed on Nov. 19,2007, the content of each of which are hereby incorporated by referencein their entirety into this application

FIELD OF THE INVENTION

This invention relates to the field of disease assessment and therapy.The invention provides compositions and methods for assessing andtreating diseases, especially cancer, and particularly lung cancer.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death worldwide, and cancer,especially lung cancer, is difficult to diagnose and treat effectively.Accordingly, there is a need in the art for new compositions and methodsfor assessing and treating various cancers, particularly lung cancer.

Lung Cancer

Lung cancer is the second most prevalent type of cancer for both men andwomen in the United States and is the most common cause of cancer deathin both men and women. The five-year survival rate for lung cancercontinues to be poor at only about 8-15%. This low survival is becauselung cancer is commonly not detected until it has spread beyond thelungs. Only 16% of new lung cancer cases in the United States aredetected at the earliest stage, when the cancer is still localized tothe lungs. At this early stage, survival is considerably higher, withestimates as high as 70-80%. Therefore, procedures for detecting lungcancer are of critical importance to the outcome of a patient sincethese procedures have the potential to reduce mortality. Thus, there isa need for new diagnostic compositions and methods that are moresensitive and specific for detecting early lung cancer.

Furthermore, there is also a need for new diagnostic compositions andmethods for determining the stage of a patient's disease. Stagedetermination has potential prognostic value and provides criteria fordesigning optimal therapy. Biomarkers that are indicative of differentstages of lung cancer would be useful to facilitate the staging of lungcancer.

Lung cancer patients are typically monitored following initial therapyand during adjuvant therapy to determine their response to therapy andto detect persistent or recurrent disease or metastasis. Thus, there isclearly a need for lung cancer markers that are more sensitive andspecific in detecting lung cancer, its recurrence, and progression.

Although imaging modalities, such as computed tomography (CT) screening,are being studied to aid in the early detection of lung cancer,controversy remains as to the ability of these methods to impactmortality (I-ELCAP Investigators, NEJM 2006 (355):1763-71 and Bach etal. 2007. JAMA 297:953-961). In addition, the most advanced imagingtechnologies under study are expensive and not widely available. TheseCT imaging tests may lead to over-diagnosis of lung cancer, resulting insignificant expenses to the health care system to manage patients withpulmonary nodules observed through these CT imaging tests. Furthermore,there is significant morbidity associated with the management of thepulmonary nodules in an effort to ascertain whether the nodules aremalignant or benign. It is estimated that 10-50% of smokers in a highrisk group have pulmonary nodules upon imaging studies (CHEST 2007Supplement—for the Treatment of Patients With Pulmonary Nodules: When IsIt Lung cancer?: AACP Evidence-Based Clinical Practice Guidelines).Thus, there is a significant need for novel diagnostics that can be usedeither independently or with imaging modalities for early diagnosis andimproved management of patients with lung cancer. For example, a bloodtest for biomarkers that has high performance (e.g., high sensitivityand specificity) for detecting lung cancer could provide a low costcomplement to CT testing for early detection of cancer. If theperformance of a biomarker test were sufficiently high, such a testcould serve as a lower cost alternative to CT or X-ray testing. Forexample, only those patients that tested positive in a biomarker testmay then need to undergo more expensive imaging tests. Furthermore, abiomarker test could be used, for example, in a yearly screening regimenfor lung cancer.

Although there have been reports of circulating tumor markers andantigens with potential use in lung cancer (see Schneider, J. 2006.Advances in Clin Chem, 42: 1-41 for a review), markers currently usedgenerally suffer from low sensitivity and less than desirablespecificity, especially among smokers (Schneider, 2006), and aretypically only used to monitor for recurrence of lung cancer. Thus,there is a need in the art for a panel of markers with high sensitivity(and varying specificities, depending on the clinical indication), suchas for detecting lung cancer. Furthermore, there is also a need fornovel markers that are useful individually or as part of a panel fordetecting lung cancer. Such markers, and panels of markers, wouldfacilitate management of patients with lung cancer, for example.

For a further review of lung cancer diagnostics, including the use oftumor biomarkers as well as CT screening, see the following citations:Schneider, “Tumor markers in detection of lung cancer”, Adv Clin Chem.2006; 42:1-41; Bach et al., “Computed tomography screening and lungcancer outcomes”, JAMA. 2007 Mar. 7; 297(9):953-61; and InternationalEarly Lung Cancer Action Program Investigators et al., “Survival ofpatients with stage I lung cancer detected on CT screening”, N Engl JMed. 2006 Oct. 26; 355(17):1763-71. Also see Pepe et al., “Phases ofbiomarker development for early detection of cancer”, J Nat'l CancerInst. 2001. 93(14):1054-1061

Description of Tables 1-2

Tables 1 and 2 provide further information for lung cancer markers(“LCM”), including their names, symbols (alternative symbols areindicated in parentheses), Genbank protein accession numbers, and anexemplary protein sequence for each marker (except for the carbohydrateantigens CA 242, CA 19-9, and CA 72-4, for which representative journalcitations are provided for each). Exemplary LCM protein sequences areprovided as SEQ ID NOS:1-65 (additionally, the carbohydrate antigens CA242, CA 19-9, and CA 72-4 are also provided). Nucleic acid sequences(e.g., mRNA transcript sequences and genomic DNA) and alternativeprotein sequences for each marker are well known in the art and canreadily be derived using the information provided in Tables 1-2, forexample.

The LCM provided in Table 1 are as follows (alternative names/symbolsare indicated in parentheses): SLPI, MIF, TIMP1, TFPI, ENO2 (NSE), CEA(CEACAM5), MMP2, AMBP, Cyfra 21-1 (Cyfra, KRT19), SCC (SERPINB3), OPN,defensin (DEFA1, HNP-1, HNP1-3), CA 242, CA 19-9, CA 72-4, MN/CAIX(CA9), ProGRP (GRP), KRT18 (TPS), ECAD (CDH1), TIMP2, CD44, LGALS3BP,ERBB2 (HER-2), UPA (PLAU), DKK (DKK1), CHGA, VEGF, KITLG, PBEF(visfatin), SORT1 (sortilin), MDK (midkine), IGFBP3, IGFBP4, CTSC,ICAM3, CTGF, LCN2, EGFR, BGN, TIMP3, HGF, MUC16 (CA125), NCAM, CRP,SERPINA1 (ATT), PKM2, RBP, KLK11, KLK13, SAA, and APOC3.

The LCM provided in Table 2 (which are particularly useful asautoantibody markers) are as follows (alternative names/symbols areindicated in parentheses): TP53 (p53), KLKB1, CFL1 (CFLN), EEF1G, HSP90α(HSP90AA1), RTN4, ALDOA, GLG1, PTK7, EFEMP1, SLC3A2 (CD98), CHGB,CEACAM1, ALCAM, HSPB1 (HSP27), LGALS1, and B7H3.

Elevated levels of each of these LCM are indicative of lung cancer,except for sortilin (SORT1), for which low levels are indicative of lungcancer.

Description of Tables 3-12

Table 3 provides 35 different panels of 11 markers (each row of 11markers represents a panel) that have at least 98% specificity and 82%sensitivity for detecting lung cancer. The total number of occurrencesof each marker in these 35 11-marker panels is indicated at the bottomof Table 3. Seven markers (SLPI, TIMP1, TFPI, SCC, OPN, CEA and CA242)appear in all 35 of these panels, GRP appears in 33 of these 35 panels,MIF appears in 29 of these 35 panels, and NSE and HNP-1 each appear in15 of these 35 panels. AMBP, Cyfra, MMP2, Ca72-4, Ca19-9, and CAIX eachappear in 7-9 of these panels, as indicated in Table 3.

Table 4 provides markers the can be included in any of the panelsdisclosed herein. For example, the markers in Table 4 can be added toany of the panels disclosed herein and/or can replace one or moremembers of any of the panels disclosed herein. As a specific example,the markers in Table 4 can be added to any of the panels disclosed inTable 5 and/or can replace one or more members of any of the panelsdisclosed in Table 5. The markers disclosed in Table 4 are alsodisclosed in Table 2.

Tables 5-12 provide data for the analysis of various panels in variouslung cancer uses, such as distinguishing lung cancer samples versusnormal samples such as for diagnosing/detecting lung cancer (Tables 5-6and 11-12, for example), as well as certain specific uses (thesespecific uses, which may be referred to herein as “indications” or asdetermining or assessing lung cancer “characteristics”, are provided inTables 7-10, for example). In Tables 5-12, each row represents a panel(a panel may comprise an individual marker). For each panel in Tables5-12, data are presented based on logistic regression and/or split pointanalysis (as indicated in each table). Area under the curve (AUC),sensitivity at 95% specificity, and specificity at 95% sensitivity areindicated. “Size” (second column) indicates the number of markers in thegiven panel. Further information regarding characteristics of the samplesets (the “54×53”, “50×50”, and “44×44” sample sets) used in each of theanalyses is provided in FIG. 16 (the “104×103” sample set used in Table8 is the “54×53” and “50×50” sample sets combined). In Tables 5-12, andelsewhere herein, “trained” refers to the sample set (which may bereferred to as the “training set”) which was used to formulate cutofflevels, and “tested” refers to the sample set (which may be referred toas the “testing set”) to which these cutoff levels were applied (such asto classify a sample as a lung tumor or normal sample, or other specificuse, based on whether marker levels were above or below the cutofflevels established from the training set).

Table 5 provides data for logistic regression and split-point analysisof the 9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,OPN, and MDK, and all subcombinations thereof (including individualmarkers), in distinguishing lung tumor samples versus normal (i.e.,control/healthy) samples, such as for diagnosing/detecting lung cancer.For each panel in Table 5, data are presented based on logisticregression and split point analysis and based on analysis of eithertraining and testing on the same 54×53 (54 controls and 53 cases) sampleset, or training on the 54×53 sample set and testing on the 50×50 (50controls×50 cases) sample set (see FIG. 16 for characteristics of thesesample sets). Area under the curve (AUC), sensitivity at 95%specificity, and specificity at 95% sensitivity are indicated. Thepanels are sorted based on the AUC indicated in the third column. “Size”(second column) indicates the number of markers in the given panel.Thus, Table 5 provides the 9-marker panel of Cyfra, SLPI, TIMP1, SCC,TFPI, CEACAM5, MMP2, OPN, and MDK, and all panel subcombinationsthereof, including each of these nine markers individually (each rowrepresents a panel).

Table 6 provides data for split-point analysis of panels (includingindividual markers) that include any of the nine markers in the panelsprovided in Table 5 and/or various other markers (which are not in thepanels provided in Table 5) in distinguishing lung tumor samples versusnormal (i.e., control/healthy) samples, such as for diagnosing/detectinglung cancer.

Table 7 provides data for logistic regression analysis of the 9-markerpanel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK, andsubcombinations thereof (including individual markers), indistinguishing adenocarcinoma versus squamous cell carcinoma types oflung cancer.

Table 8 provides data for split-point analysis of the 9-marker panel ofCyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK, andsubcombinations thereof (including individual markers), indistinguishing stage I versus stage III lung cancer. In addition totheir utility in distinguishing between early and late stage lung cancer(e.g., stage I or II versus stage III or IV), the panels provided inTable 8 are also useful for distinguishing between any other stages oflung cancer (e.g., any of stages I, II, III, and IV).

Table 9 provides data for split-point analysis of various panels indistinguishing small cell lung cancer (SCLC) versus other types of lungcancer (e.g., non-small cell lung cancer, NSCLC). In the left-side ofTable 9, marker levels are higher in NSCLC (as compared to SCLC). In theright-side of Table 9, marker levels are higher in SCLC (as compared toNSCLC).

Table 10 provides data for split-point analysis of the 9-marker panel ofCyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK, andsubcombinations thereof (including individual markers), indistinguishing malignant lung tumors versus benign lung lesions.

Table 11 provides data for split-point analysis of various panels indistinguishing small cell lung cancer (SCLC) versus normal (i.e.,control/healthy) samples.

Table 12 provides data for split-point analysis of various panels indistinguishing lung cancer (including both small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC)) versus normal (i.e.,control/healthy) samples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Shows relative expression levels for exemplary lung cancermarkers (LCM) screened by ELISA in a sample set of 12 lung tumor and 12normal serum samples. The left portion of the table shown in FIG. 1provides tumor samples identified by histology and tumor state(histology abbreviations for tumor samples are “AS”=adenosquamous,“A”=adenocarcinoma, “SC”=squamous cell carcinoma, and“BAC”=bronchioalveolar carcinoma), and the right portion of the tableshows normal samples (identified as “N” for histology). The table isbased on mean concentration values of each sample and uses 2 standarddeviations (2SD) above normal mean as the cutoff; the value is expressedas fold change from normal mean (thus, any fold change with 2SD abovenormal mean is above the cutoff). The column labeled “CRA MS” is asummary of mass spectrometry data that indicates the number ofdifferentially expressed lung tumor samples and the median mass specratio of these samples (numerical representation of over-expression isindicated by 2.0 or more, whereas numerical representation ofunder-expression is indicated by 0.5 or less) (lung tumor sampleabbreviations for mass spectrometry are “CL LU”=lung cancer cell lines,“TS LU”=lung cancer tissues, and “CM LU”=lung cancer conditionedmedium).

FIG. 2. Shows relative expression levels based on ELISA screening of asample set of 12 lung tumor (upper section) and 12 normal (lowersection) serum samples for the eight markers TFPI, SCC (interchangeablyreferred to as SSC), CEA, CA242, MNCAIX, OPN, Cyfra 21-1, and MIF (asalso shown in FIG. 1). The table is based on mean concentration valuesof each sample and uses 2 standard deviations (2SD) above normal mean asthe cutoff; the value is expressed as fold change from normal mean(thus, any fold change with 2SD above normal mean is above the cutoff).Any value below the cut-off is recoded as 0. Any or all of these eightmarkers may be used in combination as a panel for lung cancerassessment, and the panel may optionally include additional markers.

FIG. 3. Shows the performance of the eight marker panel of TFPI, SCC,CEA, CA242, MNCAIX, OPN, Cyfra 21-1, and MIF. Using an algorithm inwhich markers greater than or equal to two standard deviations werescored “positive”, this panel of eight markers had a sensitivity of 92%and specificity of 100% among the 12 sera from lung cancer patients and12 sera from healthy controls (“FP”=false positives, “TP”=truepositives, “FN”=false negatives, and “TN”=true negatives)

FIG. 4. Shows relative expression levels based on ELISA screening of asample set of 12 lung tumor (left portion; histology (“Hist”)abbreviations are “AS”=adenosquamous, “A”=adenocarcinoma, “SC”=squamouscell carcinoma, “OC”=oat cell carcinoma, and “BAC”=bronchioalveolarcarcinoma) and 12 normal (right portion; identified as “N” forhistology) serum samples for alternate panels of LCM, including a panelof the markers SLPI, TFPI, OPN, MIF, TIMP1, and MMP2. Any or all ofthese markers can also be used in any combination with any or all of thefollowing markers: CA242, SCC, CEA, NSE, CA72-4, CA19-9, Cyfra 21-1, andMN/CAIX, as shown in FIG. 4. The table is based on mean concentrationvalues of each sample and uses two standard deviations (2SD) abovenormal mean as the cut-off; the value is expressed as fold change fromnormal mean (thus, any fold change with 2SD above normal mean is abovethe cutoff). Any value below the cut-off is recoded as 0.

FIG. 5. Shows scatter plots of ELISA data for the six markers CEA, TFPI,MIF, TIMP1, OPN, and Cyfra 21-1 in 44 normal and 44 lung tumor samples,with exemplary cut-offs indicated (dotted lines). Cut-offs can beapplied that maximize sensitivity while not compromising specificity ofthe panel, for example.

FIG. 6. Shows results of ELISA analysis for the 11 markers Cyfra 21-1,MIF, TIMP1, TFPI, CEA, OPN, SCC, SLPI, HNP-1, GRP, and CA242 in 39control (normal) samples (left portion, labeled “Control”) and 39 lungtumor samples (right portion, labeled “Tumor”). Values shown areconcentration (ng/mL). Manually defined cut-offs are indicatedimmediately below each marker name. The columns labeled “#>cutoff”indicate the total number of markers with elevated expression (i.e., aconcentration greater than the manually defined cut-offs) in a givenserum sample. “Stage” indicates lung cancer stage, and “Hist Type”indicates histology type. Any or all of these 11 markers may be used incombination as a panel for lung cancer assessment, and the panel mayoptionally include additional markers.

FIG. 7. Shows performance of exemplary panels of markers, demonstratingthat increased sensitivity can be achieved by including additionalmarkers. The marker CEA provides 55% sensitivity and 90% specificity,the two markers CEA and OPN provide 60% sensitivity and 90% specificity,the three markers TFPI, CEA, and OPN provide 67% sensitivity and 90%specificity, and the four markers TIMP1, TFPI, CEA, and OPN provide 69%sensitivity and 90% specificity. The score is the sum of the log 2 ofthe ratios of the tumor concentration to the mean concentration innormal serum. ROC curves can be constructed by varying the cut-off ofthe score needed to call a sample a tumor.

FIG. 8. Shows examples of applying a cut-off for various markers (Cyfra21-1, MIF, SLPI, TIMP1, SCC, NSE, TFPI, CEA, MMP2, OPN, and AMBP areshown) that provides desirable performance for that marker. The circlesshow the approximate location of an exemplary cut-off for each markerwhich is the point on the curve that is closest to the upper-leftcorner. Different criteria can also be used, for instance falsenegatives could be weighted more heavily than false positives.

FIGS. 9-10. Shows AUC (area under curve)=0.8543 for markers MIF, TIMP1,TFPI, CEA, and OPN (FIG. 9), and AUC=0.8518 for markers TIMP1, NSE, CEA,and OPN. Score is the number of markers greater than the cutoff thatbest separates tumor samples from normal samples for each marker. ROCcurve can be constructed by varying the cut-off of the score needed tocall a sample a tumor.

FIG. 11. Shows results of analysis for certain autoantibody markers(bottom table), as well as certain other lung cancer markers (toptable). In the bottom table (autoantibody markers), the column labeled“Lung MS data” indicates a summary of where differential expression hasbeen observed by mass spectrometry (CL=cell lines, TS=tissues,CM=conditioned medium, and IP=immunoprecipitation), the column labeled“SEREX data” indicates autoantibody markers that overlap with theSerological Expression (SEREX) database which identifies markers thatelicit a high-titer IgG antibodies, and the column labeled “Rec Protein”indicates the source of recombinant protein used for autoantibodyanalysis (“vendor” indicates an external commercial source and “CRA”indicates an internal source). Histology abbreviations for tumor samplesin the top table are “AS”=adenosquamous, “A”=adenocarcinoma,“SC”=squamous cell carcinoma, and “SM”=small cell carcinoma.

FIG. 12. Shows exemplary autoantibody LCM, which can be used alone or incombination with other LCM. Certain of these autoantibody LCM are alsoprovided in Table 2 along with other autoantibody LCM.

FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D. Shows autoantibody responsesobserved in lung cancer and normal serum samples for the autoantibodymarkers KLKB1 (FIG. 13B), cofilin (FIG. 13C), and LGALS1 (FIG. 13D), aswell as p53 (FIG. 13A). Along the horizontal axis, T1 through T12indicate tumor samples and N1 through N12 indicate normal samples.

FIG. 14. Shows autoantibody detection in lung cancer and normal serumsamples for the autoantibody markers CA12, KLKB1, CFLN, LGALS1, andEEF1G, as well as p53. Autoantibody responses were detected for CA12,KLKB1, and CFLN (cofilin). The table is based on mean concentrationvalues of each sample and uses 2SD above normal mean as the cut-off. Anyvalue below the cutoff is recoded as 0. p53 showed 0 response in normalsera, therefore absolute titers are listed for p53 (positiveantibody-dependent values).

FIG. 15. Shows three additional LCM: visfatin (PBEF), sortilin (SORT-1),and midkine (MDK). Any or all of these three LCM can be implemented in apanel of markers for lung cancer diagnosis, for example. FIG. 15 showsabundance levels (in ng/mL) of these three markers in 12 normal lung and12 lung tumor samples based on ELISA analysis. For sortilin (SORT-1),abundance levels (by relative copy number) of this marker based on mRNAexpression analysis of 22 normal lung and 23 lung tumor samples is alsoprovided. For sortilin, lung tumor samples have a decreased abundancelevel of this marker compared with normal lung samples.

FIG. 16. Shows clinicopathological characteristics of lung cancer serumsamples used in various analyses disclosed herein.

FIG. 17. Shows results of ELISA analysis for the 9-marker panel ofCyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK in 50 control(normal) samples (left portion, labeled “Normal”) and 50 lung tumorsamples (right portion, labeled “Tumor”), using split-point analysisapplying manually defined cut-offs. The manually defined cut-offs areindicated immediately below each marker name. Values shown areconcentration (ng/mL). The columns labeled “≥cut off” indicate the totalnumber of markers with elevated expression (i.e., a concentrationgreater than or equal to the manually defined cut-offs) in a given serumsample. “Histology” indicates histology type (“adeno”=adenocarcinoma,“squ”=squamous cell carcinoma, “nsm” or “n-sm”=non-small cell carcinoma,“bro”=bronchioloalveolar carcinoma, “LG”=large cell carcinoma, and“neuro”=neuroendocrine). Any or all of these nine markers may be used incombination as a panel for lung cancer assessment, and the panel mayoptionally include additional markers.

FIG. 18. Describes the analysis of markers to monitor lung tumorregression/recurrence, with CEA and Cyfra as examples. In particular,levels of biomarkers in patient serum 2-4 weeks following surgery werecompared to pre-surgical marker levels.

FIG. 19. Shows an analysis of markers to monitor for lung tumorregression/recurrence. Percentage change in levels of biomarkers inpatient serum 2-4 weeks post-surgery as compared to pre-surgical levelsis indicated.

FIG. 20. Shows an analysis of the expression levels of the 9-markerpanel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK inthe following co-morbid lung diseases: asthma, bronchitis, and benignlung diseases. Values shown are concentration (ng/mL). The columnlabeled “#>cut off” indicates the total number of markers with elevatedexpression (i.e., a concentration greater than the manually definedcut-offs) in a given serum sample. The manually defined cut-offs areindicated immediately below each marker name (in the row labeled“Cut-off”).

FIG. 21. Shows an analysis of integrating an exemplary supplementalbiomedical parameter (smoking history) with an exemplary LCM panel(TIMP1, TFPI, CEACAM5, and Ca72-4) plus pack years (“pack year”: numberof cigarettes smoked per day multiplied by number of years of smoking atthis rate). The left-side graph shows that performance of a 5-markerpanel (represented by the line that includes a vertical portion at 50 ofthe x-axis) is enhanced with addition of smoking history (pack years)(represented by the line that includes a vertical portion at about 43 ofthe x-axis). Sensitivity increases from 71.5% to 84.6%. The right-sidegraph shows split-point analysis performed following the addition ofsmoking history (split at about 45 of the x-axis).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The invention will best be understood by reference to the followingdetailed description of the exemplary embodiments, taken in conjunctionwith the accompanying table(s) and/or figure(s). The discussion below isexemplary and is not to be taken as limiting the scope defined by theclaims.

Exemplary embodiments of the invention provide the following markers(see Tables 1-2), combinations of these markers, and methods of usingthese markers, particularly for lung cancer-related uses, and especiallyfor lung cancer diagnostics (alternative names/symbols are indicated inparentheses): SLPI, MIF, TIMP1, TFPI, ENO2 (NSE), CEA (CEACAM5), MMP2,AMBP, Cyfra 21-1 (Cyfra, KRT19), SCC (SERPINB3), OPN, defensin (DEFA1,HNP-1, HNP1-3), CA 242, CA 19-9, CA 72-4, MN/CAIX (CA9), ProGRP (GRP),KRT18 (TPS), ECAD (CDH1), TIMP2, CD44, LGALS3BP, ERBB2 (HER-2), UPA(PLAU), DKK (DKK1), CHGA, VEGF, KITLG, PBEF (visfatin), SORT1(sortilin), MDK (midkine), IGFBP3, IGFBP4, CTSC, ICAM3, CTGF, LCN2,EGFR, BGN, TIMP3, HGF, MUC16 (CA125), NCAM, CRP, SERPINA1 (ATT), PKM2,RBP, KLK11, KLK13, SAA, APOC3, TP53 (p53), KLKB1, CFL1 (CFLN), EEF1G,HSP90α (HSP90AA1), RTN4, ALDOA, GLG1, PTK7, EFEMP1, SLC3A2 (CD98), CHGB,CEACAM1, ALCAM, HSPB1 (HSP27), LGALS1, and B7H3, which are collectivelyreferred to herein as “LCM” (“lung cancer markers”). Elevated levels ofeach of these LCM are indicative of lung cancer, except for sortilin(SORT1), for which low levels are indicative of lung cancer. Tables 1and 2 provide further information for each of these LCM, including theirnames, symbols, Genbank protein accession numbers, and an exemplaryprotein sequence for each marker (except for the carbohydrate antigensCA 242, CA 19-9, and CA 72-4, for which representative journal citationsare provided for each). Exemplary LCM protein sequences are provided asSEQ ID NOS:1-65 (additionally, the carbohydrate antigens CA 242, CA19-9, and CA 72-4 are also provided). Nucleic acid sequences (e.g., mRNAtranscript sequences and genomic DNA) and alternative protein sequencesfor each marker are well known in the art and can readily be derivedusing the information provided in Tables 1-2, for example. The markersprovided in Table 2 are particularly useful as autoantibody markers.

Certain embodiments of the invention provide combinations comprising,consisting of, and consisting essentially of the following nine LCM, andsubcombinations thereof (these nine LCM may be referred to herein as the“9-marker panel”, which is shown in FIG. 17 and Table 5, for example):Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK. Certainembodiments of the invention provide compositions based on this 9-markerpanel and subcombination thereof, and methods of using this 9-markerpanel, particularly for uses related to lung cancer (such as detectinglung cancer). In certain embodiments, one or more members of this9-marker panel is replaced by one or more markers shown in Table 4and/or one or more markers shown in Table 4 is added to this 9-markerpanel. With respect to the nine markers Cyfra, SLPI, TIMP1, SCC, TFPI,CEACAM5, MMP2, OPN, and MDK, elevated levels are indicative of lungcancer (for all of the LCM disclosed herein, elevated levels areindicative of lung cancer, except for sortilin (SORT1), for which lowlevels are indicative of lung cancer). In certain embodiments, if thelevels of two or more markers (i.e., a “plurality”) of the 9-markerpanel are elevated in a sample (e.g., a serum sample) from anindividual, this indicates that the individual has lung cancer. Invarious other embodiments, if the levels of one or more, three or more,four or more, five or more, six or more, seven or more, eight or more,or all nine markers of the 9-marker panel are elevated in a sample froman individual, this indicates that the individual has lung cancer. Incertain embodiments, a marker is classified as being elevated if itslevel is greater than (or greater than or equal to) a predeterminedcutoff level.

Furthermore, certain embodiments of the invention provide combinationscomprising, consisting of, and consisting essentially of the followingsix LCM (each of which is also contained in the above 9-marker panel),and subcombinations thereof (these six LCM may be referred to herein asthe “6-marker subset of the 9-marker panel”, which is shown in Table 5):Cyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK. Certain embodiments of theinvention provide compositions based on this 6-marker subset of the9-marker panel and subcombination thereof, and methods of using this6-marker subset of the 9-marker panel, particularly for uses related tolung cancer (such as detecting lung cancer). In certain embodiments, oneor more members of this 6-marker subset of the 9-marker panel isreplaced by one or more markers shown in Table 4 and/or one or moremarkers shown in Table 4 is added to this 6-marker subset of the9-marker panel. With respect to the six markers Cyfra, SLPI, TIMP1,TFPI, CEACAM5, and MDK, elevated levels are indicative of lung cancer(for all of the LCM disclosed herein, elevated levels are indicative oflung cancer, except for sortilin (SORT1), for which low levels areindicative of lung cancer). In certain embodiments, if the levels of twoor more markers (i.e., a “plurality”) of the 6-marker subset of the9-marker panel are elevated in a sample (e.g., a serum sample) from anindividual, this indicates that the individual has lung cancer. Invarious other embodiments, if the levels of one or more, three or more,four or more, five or more, or all six markers of the 6-marker subset ofthe 9-marker panel are elevated in a sample from an individual, thisindicates that the individual has lung cancer. In certain embodiments, amarker is classified as being elevated if its level is greater than (orgreater than or equal to) a predetermined cutoff level.

Exemplary embodiments of the invention provide LCM and combinations ofLCM (combinations of LCM may be interchangeably referred to herein aspanels), and uses thereof, particularly uses related to lung cancer. Forexample, exemplary embodiments of the invention provide methods andcompositions for assessing (e.g., diagnosing/detecting, prognosing, orpredicting drug response), treating, and preventing diseases, especiallycancer, and particularly lung cancer, using LCM. Furthermore, thecompositions and methods of the invention may be suitable for othertypes of cancer, particularly other epithelial cell-related cancers andsolid tumors, as well as other lung diseases.

LCM proteins and fragments thereof (LCM peptides), LCM carbohydrateantigens and fragments thereof, and LCM nucleic acid molecules andfragments thereof encoding LCM proteins and peptides, are collectivelyreferred to as “LCM” or “markers” (which may be interchangeably referredto as “biomarkers”, “antigens”, or “targets”).

The terms “protein” and “polypeptide” are used herein interchangeably.Furthermore, references herein to proteins/polypeptides may alsotypically encompass carbohydrate antigens (“CA”); for example,references to LCM proteins/polypeptides may also typically encompass thecarbohydrate antigens CA 242, CA 19-9, and CA 72-4. Exemplary LCMprotein/polypeptide sequences are provided as SEQ ID NOS:1-65(additionally, carbohydrate antigens CA 242, CA 19-9, and CA 72-4 arealso provided). A “peptide” typically refers to a fragment of aprotein/polypeptide. Thus, peptides are interchangeably referred to asfragments. References herein to proteins, peptides, carbohydrateantigens, nucleic acid molecules, and antibodies typically are notlimited to the full-size or full-length molecule, but also can encompassfragments of these molecules (unless a particular sequence or structureis explicitly stated).

As used herein, a “lesion” (e.g., a lung lesion) may be interchangeablyreferred to as a “nodule” (e.g., a lung nodule), and “lung” may beinterchangeably referred to as “pulmonary”.

As used herein, “subcombinations” (of LCM) may be interchangeablyreferred to as “subsets” (of LCM).

“Abundance level” may be interchangeably referred to herein as“expression level”, or just “level” or “abundance”. Determination of LCMlevels may be referred to herein as “quantifying” LCM, or“quantification” of LCM.

A “differential” abundance level is a level of a marker (e.g., LCMprotein or nucleic acid) in a test sample (e.g., a disease sample)either above or below the normal abundance level of the same marker in acorresponding control or normal sample or group of control/normalsamples (e.g., a sample set or population). Thus, for example, a“differential” abundance level can encompass either a “high” (or“increased”) or “low” (or “decreased”) abundance level. An example of anormal abundance level for a LCM is the mean abundance level of themarker in individuals who do not have lung cancer, which may be the meanabundance of the marker in, for example, a particular control sample setor population of individuals who do not have lung cancer. The normalabundance may also be the typical abundance level of a marker in anormal cell (e.g., a normal lung cell) compared with the typicalabundance level of the marker in a corresponding disease cell (e.g., alung cancer cell).

An example of a “high”, “increased”, or “elevated” (these terms are usedherein interchangeably) abundance level for a LCM is an abundance levelthat is at least two standard deviations above the normal abundancelevel of the marker (e.g., the mean abundance level of the marker inindividuals who do not have lung cancer). An example of a “low” or“decreased” abundance level for a LCM is an abundance level that is atleast two standard deviations below the normal abundance level of themarker (e.g., the mean abundance level of the marker in individuals whodo not have lung cancer). Thus, in this particular example, an abundancelevel that is between 2 standard deviations above and 2 standarddeviations below the mean abundance level of the marker in individualswho do not have lung cancer may be considered within a normal abundancelevel range. These are merely exemplary cut-offs which can be used tolabel an abundance level of a marker as “high”/“increased” or“low”/“decreased”.

In alternative exemplary embodiments, the cut-offs for a“high”/“increased” or “low”/“decreased” abundance can be an abundancelevel that is greater that one standard deviation above or below thenormal abundance level, or greater that three standard deviations aboveor below the normal abundance level, or any other desired standarddeviation. In further alternative exemplary embodiments, the cut-offsfor a “high”/“increased” or “low”/“decreased” abundance can be baseddirectly on the expression ratio or fold difference, for example, a2-fold increase/decrease, 3-fold increase/decrease, or 4-foldincrease/decrease, or any other desired degree of increase/decrease.Further, the normal abundance level can be based on, for example, eitherthe mean or median abundance level (e.g., of a given control sampleset). Other exemplary methods for developing cut-offs for“high”/“increased” or “low”/“decreased” abundance levels includedetermining a normal abundance level range (such as by testing a panelof markers in a control sample set of normal lung tissue samples), andclassifying any test samples above or below this normal range (orabove/below a desired threshold relative to this normal range, such asoutside a particular percentage of samples within this normal range suchas above or below 95% of samples within the normal range) as“high”/“increased” or “low”/“decreased”, respectively.

A wide variety of further cut-offs for classifying the abundance levelof a marker as “high”/“increased” or “low”/“decreased”, and methods forformulating these cut-offs, are known in the art and/or can beimplemented by one of ordinary skill in the art. For a given marker orpanel of markers, various cut-offs can be applied, such as cut-offs thatmaximize sensitivity while maintaining a desired specificity, forexample, or that maximize specificity while maintaining a desiredsensitivity. For example, the classification of a sample as a tumorsample or normal sample can be accomplished using a variety of methodsthat may involve using a set of training data to produce a model thatcan then be used to classify a test sample (such as to diagnose lungcancer, for example). Tumor/normal cut-offs can be selected by manualinspection of multiple markers from the training data set, and thesecut-offs can be applied to classifying test samples (such as tocharacterize patient samples with respect to lung cancer). Exemplarymethods include, but are not limited to, split-point analysis (e.g., Moret al., “Serum protein markers for early detection of ovarian cancer”,Proc Natl Acad Sci USA. 2005 May 24; 102(21):7677-82, incorporatedherein by reference), logistic regression analysis (e.g., Planque etal., “A multiparametric serum kallikrein panel for diagnosis ofnon-small cell lung carcinoma”, Clin Cancer Res. 2008 Mar. 1;14(5):1355-62, incorporated herein by reference), Naïve Bayes,multivariate analysis, decision tree modeling (e.g., Patz et al., “Panelof serum biomarkers for the diagnosis of lung cancer”, J Clin Oncol(2007), 25, 5578-5583), and other classification methods (see, forexample, Dudoit et al., “Classification in Microarray Experiments”,Statistical Analysis of Gene Expression Microarray Data, 2003, Chapman &Hall/CRC: 93-158, incorporated herein by reference).

The terms “sensitivity” and “specificity” are used herein with respectto the ability of one or more markers to correctly classify a sample asa tumor sample or a non-tumor sample (a non-tumor sample may beinterchangeably referred to as a “normal”, “control”, or “healthy”sample), respectively. “Sensitivity” indicates the performance of themarker(s) with respect to correctly classifying tumor samples.“Specificity” indicates the performance of the marker(s) with respect tocorrectly classifying non-tumor samples. For example, 98% specificityand 85% sensitivity for a panel of markers used to test a set of controland tumor samples indicates that 98% of the control samples werecorrectly classified as control samples by the panel, and 85% of thetumor sample were correctly classified as tumor samples by the panel.

Area under the curve (AUC) refers to the area under the curve of areceiver operating characteristic (ROC) curve, which are well known inthe art (see, e.g., Planque et al., “A multiparametric serum kallikreinpanel for diagnosis of non-small cell lung carcinoma”, Clin Cancer Res.2008 Mar. 1; 14(5):1355-62, incorporated herein by reference). AUCmeasures are useful for comparing the accuracy of a classificationalgorithm across the complete data range. Classification algorithms witha greater AUC have a greater capacity to classify unknowns correctlybetween two groups of interest (e.g., lung cancer samples and normalsamples). ROC curves are useful for plotting the performance of aparticular feature (e.g., an LCM and/or a supplemental biomedicalparameter) in distinguishing between two populations (e.g., cases havinglung cancer and controls without lung cancer). Typically, the featuredata across the entire population (e.g., the cases and controls) aresorted in ascending order based on the value of a single feature. Then,for each value for that feature, the true positive and false positiverates for the data are calculated. The true positive rate is determinedby counting the number of cases above the value for that feature andthen dividing by the total number of cases. The false positive rate isdetermined by counting the number of controls above the value for thatfeature and then dividing by the total number of controls. Although thisdefinition refers to scenarios in which a feature is elevated in casescompared to controls, this definition also applies to scenarios in whicha feature is lower in cases compared to the controls (in such ascenario, samples below the value for that feature would be counted).ROC curves can be generated for a single feature as well as for othersingle outputs, for example, a combination of two or more features canbe mathematically combined (e.g., added, subtracted, multiplied, etc.)to provide a single sum value, and this single sum value can be plottedin a ROC curve. Additionally, any combination of multiple features, inwhich the combination derives a single output value, can be plotted in aROC curve. These combinations of features may comprise a test. The ROCcurve is the plot of the true positive rate (sensitivity) of a testagainst the false positive rate (specificity) of the test.

Exemplary embodiments of the invention, which are discussed in greaterdetail below, provide antibodies, proteins, carbohydrate antigens,immunogenic peptides (e.g., peptides which induce a T-cell response), orother biomolecules, as well as small molecules, nucleic acid agents(e.g., RNAi and antisense nucleic acid agents), and other compositionsthat modulate the markers (e.g., agonists and antagonists), such as bybinding to or otherwise interacting with or affecting the markers. Thesecompositions can be used for assessing, treating, and preventingdiseases, especially cancer, and particularly lung cancer, as well asother uses. Moreover, the invention provides methods for assessing,treating, and preventing diseases such as lung cancer, particularly byusing these compositions. Further provided are methods of screening foragents that modulate LCM, such as by affecting the function, activity,and/or expression level of LCM, and agents identified by these screeningmethods.

Exemplary embodiments of the invention also provide methods ofmodulating cell function, especially lung cell function. In particular,the invention provides methods of modulating cell proliferation and/orapoptosis. For example, for cancer/tumor cells, the invention providesmethods of inhibiting cell proliferation and/or stimulating apoptosis.Such methods can be applied to the treatment of diseases, especiallycancer, and particularly lung cancer. In certain exemplary embodiments,the invention provides methods of treating lung cancer by targeting LCMto thereby inhibit proliferation of lung cancer cells and/or stimulateapoptosis of lung cancer cells.

Exemplary embodiments of the invention further provide methods ofdetermining or predicting effectiveness or response to a particulartreatment, and methods of selecting a treatment for an individual,particularly a lung cancer treatment. For example, markers that aredifferentially expressed by cells (e.g., lung cancer cells) that aremore or less responsive (sensitive) or resistant to a particulartreatment, such as a cancer treatment, are useful for determining orpredicting effectiveness or response to the treatment or for selecting atreatment for an individual.

Exemplary embodiments of the invention also provide methods of selectingindividuals for a clinical trial of a therapeutic agent, particularly aclinical trial for lung cancer or other cancer. For example, the markerscan be used to identify individuals for inclusion in a clinical trialwho are more likely to respond to a particular therapeutic agent.Alternatively, the markers can be used to exclude individuals from aclinical trial who are less likely to respond to a particulartherapeutic agent or who are more likely to experience toxic or otherundesirable side effects from a particular therapeutic agent.Furthermore, such individuals who are determined to be less likely torespond to a particular therapeutic agent can be selected for inclusionin a clinical trial of a different therapeutic agent that maypotentially benefit them.

In certain exemplary embodiments, the various individual LCM and LCMpanels described herein are provided as compositions. For example, incertain embodiments, each of the members of an LCM panel, and/orreagents for detecting each of these members, are provided as individualcompositions, such as in the form of reagents for detecting each memberof an LCM panel by ELISA assays (which may be referred to herein as“ELISA reagents”). Furthermore, in certain embodiments, compositionsthat comprise multiple members of a panel or an entire panel (and/orreagents for detecting each of these multiple members), are provided,such as in the form of kits that contain reagents (such as ELISAreagents) for detecting multiple members of a panel or an entire panel.Other compositions of the invention include arrays or other platformsthat have multiple LCM, or multiple reagents (e.g., antibodies) fordetecting multiple LCM, coupled to a substrate. In various compositionsof the invention, the LCM, or reagents for detecting LCM (e.g.,antibodies), are labeled with a detectable moiety (such as a fluorescentlabel).

Exemplary LCM Combinations/Panels

For example, using a panel of sera from 12 lung cancer patients and 12healthy control individuals, a group of 8 markers made up of TFPI, SCC,CEA, CA242, MN/CAIX, OPN, Cyfra 21-1, and MIF (FIG. 2) detected all thecancer samples except a bronchioalveolar cancer sample (which isbiologically distinct from other samples in the panel), and only a fewof these markers were detected at levels above the threshold in thehealthy control samples. When a simple algorithm was applied (i.e.,markers greater than or equal to two standard deviations were scored“positive”, using the criterion stated above), this group of eightmarkers had a sensitivity of 92% and specificity of 100% among the 12sera from lung cancer patients and 12 sera from healthy controls (nofalse positives, 11 true positives, 1 false negative, and 12 truenegatives) (FIG. 3).

An alternate panel was configured that was made up of following markers:SLPI, TFPI, OPN, MIF, TIMP1, and MMP2 (FIG. 4). Any or all of thesemarkers can also be used in any combination with any or all of thefollowing markers: CA242, SCC, CEA, NSE, CA72-4, CA19-9, Cyfra 21-1, andMN/CAIX (FIG. 4).

Further, a six-marker panel made-up of Cyfra 21-1, TIMP-1, MIF, TFPI,CEA, and OPN was also configured (FIG. 5). This six-marker panel, whentested on a larger group of 44 lung tumor sera and 44 normal sera,resulted in 75% sensitivity at 95% specificity.

Further, an 11-marker panel made-up of Cyfra, MIF, TIMP1, TFPI, CEA,OPN, SCC, SLPI, HNP-1, GRP, and CA242 was also configured (FIG. 6). This11-marker panel, when tested on a group of 39 lung tumor sera and 39normal sera, resulted in 98% specificity for controls (38/39 controls)and 85% sensitivity for tumor sera (33/39 tumors) (FIG. 6).

Table 3 shows further examples of various 11-marker panels.Specifically, Table 3 provides 35 different panels of 11 markers (eachrow of 11 markers represents a panel) that have at least 98% specificityand 82% sensitivity for detecting lung cancer. Seven markers (SLPI,TIMP1, TFPI, SCC, OPN, CEA and CA242) appear in all 35 of these panels,GRP appears in 33 of the 35 panels, MIF appears in 29 of the 35 panels,and NSE and HNP-1 each appear in 15 of the 35 panels. AMBP, Cyfra, MMP2,Ca72-4, Ca19-9, and CAIX each appear in 7-9 of the panels, as indicatedin Table 3.

Further, a 9-marker panel made-up of Cyfra, SLPI, TIMP1, SCC, TFPI,CEACAM5, MMP2, OPN, and MDK was also configured (e.g., FIG. 17 and Table5). This 9-marker panel demonstrated 98% specificity (49/50 controls)and 96% sensitivity (48/50 tumors) (FIG. 17). Additionally, a 6-markersubset of this 9-marker panel was also configured that was made-up ofCyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK (Table 5).

Other markers, which are also referred to herein as LCM and which may beused either alone or in combination with any of the other LCM describedherein in any combination, include a group of antigens to which“self-made” or “autoantibodies” are often found in the circulation ofpatients with various diseases, particularly cancer (Table 2 and FIGS.11-14). Examples of these autoantibody markers include the following:KLKB1, CFL1, LGAGS1, EEF1G, RTN4, ALDOA, HSPCA, PABPC4, NAGK, CFHL1,CSF1R, and RANBP2 (FIG. 12), and other autoantibody markers as shown inTable 2. Detection of autoantibody LCM such as these may complementother LCM and enhance the performance of LCM panels, particularly forassessing lung cancer.

The following are exemplary panels of LCM. Various exemplary embodimentsof the invention provide, for example, compositions based on thesepanels and methods of using these panels, particularly for uses relatedto lung cancer such as diagnosis of lung cancer (e.g., differentiallevels, such as elevated or low levels as compared to control/normallevels, of a plurality of markers in a panel, or all markers in panel,can indicate the presence of lung cancer). These exemplary panels mayconsist of, consist essentially of, or comprise the followingcombinations of markers:

-   -   1) Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK        (which may be referred to herein as the “9-marker panel” and is        shown in FIG. 17 and Table 5).    -   2) Cyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK (which may be        referred to herein as the “6-marker subset of the 9-marker        panel” and is shown in Table 5).    -   3) Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK        (the “9-marker panel”), which may optionally be in combination        with one or more other markers (which may be added to this        9-marker panel and/or replace one or more members of this        9-marker panel), wherein these other markers may optionally be        selected from the group consisting of the markers shown in        Tables 1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens CA        242, CA 19-9, and CA 72-4), Table 4, FIG. 1, and FIG. 12,        particularly those markers that are shown in Table 4.    -   4) Cyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK (the “6-marker        subset of the 9-marker panel”), which may optionally be in        combination with one or more other markers (which may be added        to this 6-marker subset of the 9-marker panel and/or replace one        or more members of this 6-marker subset of the 9-marker panel),        wherein these other markers may optionally be selected from the        group consisting of the markers shown in Tables 1-2 (SEQ ID        NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9, and CA        72-4), Table 4, FIG. 1, and FIG. 12, particularly those markers        that are shown in Table 4.    -   5) Any of the panels (which may include single markers) provided        in Table 5 (which provides the 9-marker panel and all        subcombinations thereof; each row of Table 5 represents a        different panel), which may optionally be in combination with        one or more other markers (which may be added to any panel in        Table 5 and/or replace one or more members of any panel in Table        5), wherein these other markers may optionally be selected from        the group consisting of the markers shown in Tables 1-2 (SEQ ID        NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9, and CA        72-4), Table 4, FIG. 1, and FIG. 12, particularly those markers        that are shown in Table 4.    -   6) Any of the panels provided in Table 5 or Table 6        (particularly the 9-marker panel of Cyfra, SLPI, TIMP1, SCC,        TFPI, CEACAM5, MMP2, OPN, and MDK, as well as subsets thereof,        and panels comprising this 9-marker panel or subsets thereof        that further include one or more additional markers such as        those panels set forth in Table 6), particularly for use in        methods for distinguishing lung tumor samples versus normal        (i.e., control/healthy) samples. These panels are particularly        useful for determining whether an individual has lung cancer        (i.e., detecting lung cancer), for example.    -   7) Any of the panels provided in Table 7 (particularly the        9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,        OPN, and MDK, as well as subsets thereof), particularly for use        in methods for distinguishing adenocarcinoma versus squamous        cell carcinoma. These panels are particularly useful for        determining whether an individual's lung cancer is        adenocarcinoma or squamous cell carcinoma, for example.    -   8) Any of the panels provided in Table 8 (particularly the        9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,        OPN, and MDK, as well as subsets thereof), particularly for use        in methods for distinguishing between any stages of lung cancer        (e.g., any of stages I, II, III, and IV), particularly between        early stage (stage I or II) and late stage (stage III or IV)        lung cancer, and especially between stage I and stage III lung        cancer. These panels are particularly useful for determining the        stage of an individual's lung cancer, for example.    -   9) Any of the panels provided in Table 9, particularly for use        in methods for distinguishing SCLC versus other types of lung        cancer (e.g., NSCLC). These panels are particularly useful for        determining whether an individual's lung cancer is SCLC or        NSCLC, for example.    -   10) Any of the panels provided in Table 10 (particularly the        9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2,        OPN, and MDK, as well as subsets thereof), particularly for use        in methods for distinguishing malignant lung tumors versus        benign lung lesions. These panels are particularly useful for        determining whether a lung lesion identified in an individual        (such as by CT screening) is a malignant tumor or a benign        lesion, for example.    -   11) Any of the panels provided in Table 11, particularly for use        in methods for distinguishing SCLC versus normal (i.e.,        control/healthy) samples. These panels are particularly useful        for determining whether an individual has SCLC, for example.    -   12) Any of the panels provided in Table 12, particularly for use        in methods for distinguishing lung cancer (including both SCLC        and NSCLC) versus normal (i.e., control/healthy) samples. These        panels are particularly useful for determining whether an        individual has lung cancer such as SCLC or NSCLC, for example.    -   13) Panels that include any or all of the 11 markers provided in        FIG. 19 (and subsets thereof, as well as panels comprising these        11 markers or subsets thereof that further include one or more        additional markers), particularly for use in methods of        monitoring for lung tumor regression and/or recurrence. These        panels are particularly useful for monitoring for lung tumor        regression and/or recurrence, for example.    -   14) Cyfra 21-1, MIF, TIMP1, TFPI, CEA, OPN, SCC, SLPI, HNP-1,        GRP, and CA242 (which may be referred to herein as the        “11-marker panel” and is shown in FIG. 6).    -   15) TFPI, CEA, MIF, TIMP1, OPN, and Cyfra 21-1 (which may be        referred to herein as the “6-marker panel” and is shown in FIG.        5).    -   16) TFPI, SCC, CEA, CA242, MN/CAIX, OPN, Cyfra 21-1 and MIF        (which may be referred to herein as the “8-marker panel” and is        shown in FIGS. 2-3).    -   17) TFPI, SLPI, OPN, MIF, TIMP1, and MMP2, any or all of which        can optionally be used in combination with any or all of the        following additional markers: CA242, SCC, CEA, NSE, CA724,        CA199, Cyfra 21-1, and MN/CAIX (see FIG. 4).    -   18) TFPI, TIMP1, CEA, and OPN (see FIG. 7).    -   19) TFPI, CEA, and OPN (see FIG. 7).    -   20) CEA and OPN (see FIG. 7).    -   21) TFPI, MIF, TIMP1, CEA, and OPN (see FIG. 9).    -   22) TIMP1, NSE, CEA, and OPN (see FIG. 10).    -   23) TFPI, CEA, TIMP-1, NSE, SLPI, SCC, Cyfra 21-1, and MIF, any        or all of which can optionally be in combination with any or all        of the following autoantibody markers: p53, KLKB1, LGALS1, CFLN,        EEF1G, HSP90a, RTN4, ALDOA, GLG1, PTK7, EFEMP1, CD98, CHGB,        B7H3, and CEACAM1 (see FIG. 11).    -   24) TFPI, SLPI, TFPI2, CEA, and TIMP1.    -   25) TFPI, SLPI, and TIMP1.    -   26) KLKB1 and cofilin (CFLN) (see FIG. 13B and FIG. 13C).    -   27) KLKB1, cofilin (CFLN), and CA12 (see FIG. 14).    -   28) TFPI, either alone or in combination with one or more other        markers, which may optionally be selected from the group        consisting of the markers shown in Tables 1-2 (SEQ ID NOS:1-65        and the carbohydrate antigens CA 242, CA 19-9, and CA 72-4),        Table 4, FIG. 1, and FIG. 12.    -   29) One or more markers selected from the group consisting of        defensin (DEFA1, HNP-1), ICAM3, CTGF, LCN2, biglycan, and HGF,        either alone or in combination with one or more other markers,        which may optionally be selected from the group consisting of        the markers shown in Tables 1-2 (SEQ ID NOS:1-65 and the        carbohydrate antigens CA 242, CA 19-9, and CA 72-4), Table 4,        FIG. 1, and FIG. 12.    -   30) Two or more markers selected from the group consisting of        TFPI, defensin, ICAM3, CTGF, LCN2, biglycan, and HGF, either        alone or in combination with one or more other markers, which        may optionally be selected from the group consisting of the        markers shown in Tables 1-2 (SEQ ID NOS:1-65 and the        carbohydrate antigens CA 242, CA 19-9, and CA 72-4), Table 4,        FIG. 1, and FIG. 12.    -   31) One or more markers shown in Table 1 (SEQ ID NOS:1-38 and        56-65 and the carbohydrate antigens CA 242, CA 19-9, and CA        72-4) and/or FIG. 1, in combination with one or more        autoantibody markers shown in Table 2 (SEQ ID NOS:39-55) and/or        FIG. 12.    -   32) Any of the 11-marker panels provided in Table 3 (each row of        Table 3 represents a different 11-marker panel).    -   33) SLPI, TIMP1, TFPI, SCC, OPN, CEA, and CA242, which may        optionally be in combination with GRP and/or MIF (see Table 3).    -   34) SLPI, TIMP1, TFPI, SCC, OPN, CEA, and CA242, which may        optionally be in combination with GRP and/or MIF, and which may        optionally further be in combination with HNP-1 and/or NSE (see        Table 3).    -   35) SLPI, TIMP1, TFPI, SCC, OPN, CEA, and CA242, which may        optionally be in combination with any or all of GRP, MIF, HNP-1,        and NSE, and which may optionally further be in combination with        any or all of CAIX, Ca19-9, Ca72-4, MMP2, Cyfra 21-1, and AMBP        (see Table 3).    -   36) SLPI, TIMP1, TFPI, SCC, OPN, CEA, and CA242, which may        optionally be in combination with any or all of GRP, MIF, HNP-1,        NSE, and Cyfra 21-1.    -   37) One or more markers selected from the group consisting of        visfatin, sortilin, and midkine, either alone or in combination        with one or more other markers, which may optionally be selected        from the group consisting of the markers shown in Tables 1-2        (SEQ ID NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9,        and CA 72-4), Table 4, FIG. 1, and FIG. 12.

Exemplary Uses of LCM

Certain exemplary embodiments of the invention relate to methods ofdetecting the presence of lung cancer in an individual by measuring theamounts of circulating LCM, such as in serum, by immunological methodsor other methods. These LCM are, for example, differentially expressed(over- or under-expressed) in individuals with lung cancer as comparedto individuals without lung cancer (individuals without lung cancer areinterchangeably referred to herein as “normal”, “control”, or “healthy”individuals). Detection of variation from a “normal” expression level,or differential expression, can be used for, for example, earlydiagnosis of lung cancer, distinguishing between a benign and malignantlung lesion (such as a lesion observed on a CT scan), monitoring lungcancer recurrence, or other clinical indications.

LCM may be used in a variety of clinical indications for lung cancer,including, but not limited to, detection of lung cancer (such as in ahigh-risk individual or population), characterizing lung cancer (e.g.,determining lung cancer type, sub-type, or stage) such as distinguishingbetween non-small cell lung cancer (NSCLC) and small cell lung cancer(SCLC) and/or between adenocarcinoma and squamous cell carcinoma (orotherwise facilitating histopathology), determining whether a lunglesion is a benign lesion or a malignant lung tumor, lung cancerprognosis, monitoring lung cancer progression or remission, monitoringfor lung cancer recurrence, monitoring metastasis, treatment selection,monitoring response to a therapeutic agent or other treatment,stratification of patients for computed tomography (CT) screening (e.g.,identifying those patients at greater risk of lung cancer and therebymost likely to benefit from spiral-CT screening, thus increasing thepositive predictive value of CT), combining LCM testing withsupplemental biomedical parameters such as smoking history, etc., orwith nodule size, morphology, etc. (such as to provide an assay withincreased diagnostic performance compared to CT testing or LCM testingalone), facilitating the diagnosis of a pulmonary nodule as malignant orbenign, facilitating clinical decision making once a lung cancer lesionis observed on CT (e.g., ordering repeat CT scans if the lesion isdeemed to be low risk, such as if an LCM-based test is negative, with orwithout categorization of lesion size, or considering biopsy if thelesion is deemed medium to high risk, such as if an LCM-based test ispositive, with or without categorization of lesion size), andfacilitating decisions regarding clinical follow-up (e.g., whether toimplement repeat CT scans, fine needle biopsy, or thoracotomy afterobserving a non-calcified lesion on CT). LCM testing may improvepositive predictive value (PPV) over CT screening alone. In addition totheir utilities in conjunction with CT screening, LCM can also be usedin conjunction with any other imaging modalities used for lung cancer,such as chest X-ray. Furthermore, LCM may also be useful for enablingcertain of these uses to be achieved before indications of lung cancerare detected by imaging modalities or other clinical correlates, orbefore symptoms appear.

As examples of how LCM may be useful for diagnosing lung cancer, a highor low abundance level (i.e., a “differential” abundance level) of oneor more LCM in an individual who is not known to have lung cancer mayindicate that the individual has lung cancer, thereby enabling earlydetection of lung cancer at an early stage of the disease when treatmentis most effective, perhaps before the lung cancer is detected by othermeans or before symptoms appear. An increase in the abundance of one ormore LCM during the course of lung cancer may be indicative of lungcancer progression, e.g., a lung tumor is growing and/or metastasizing(and thus a poor prognosis), whereas a decrease in the abundance of oneor more LCM may be indicative of lung cancer remission, e.g., a lungtumor is shrinking (and thus a good prognosis) Similarly, an increase inthe abundance of one or more LCM during the course of lung cancertreatment may indicate that the lung cancer is progressing and thereforeindicate that the treatment is ineffective, whereas a decrease in theabundance of one or more LCM during the course of lung cancer treatmentmay be indicative of lung cancer remission and therefore indicate thatthe treatment is working successfully. Additionally, an increase ordecrease in the abundance of one or more LCM after an individual hasapparently been cured of lung cancer may be indicative of lung cancerrecurrence. In a situation such as this, for example, the individual canbe re-started on therapy (or the therapeutic regimen modified such as toincrease dosage amount and/or frequency, if the patient has maintainedtherapy) at an earlier stage than if the recurrence of lung cancer wasnot detected until later. Furthermore, a differential abundance level ofone or more LCM in an individual may be predictive of the individual'sresponse to a particular therapeutic agent. In monitoring for lungcancer recurrence or progression, changes in LCM levels may indicate theneed for repeat imaging (e.g., repeat CT scanning), such as to determinelung cancer activity, or the need for changes in treatment.

Detection of LCM may be particularly useful following, or in conjunctionwith, lung cancer treatment, such as to evaluate the success of thetreatment or to monitor lung cancer remission, recurrence, and/orprogression (including metastasis) following treatment. Lung cancertreatment may include, for example, administration of a therapeuticagent to a patient, surgery (e.g., surgical resection of at least aportion of a lung tumor), radiation therapy, or any other type of lungcancer treatment used in the art, and any combination of thesetreatments. For example, LCM may be detected at least once aftertreatment or may be detected multiple times after treatment (such as atperiodic intervals), or may be detected both before and after treatment.A differential abundance level of LCM, such as an increase or decreasein the abundance level of LCM after treatment compared with theabundance level of LCM before treatment, or an increase or decrease inthe abundance level of LCM at a later time point after treatmentcompared with the abundance level of LCM at an earlier time point aftertreatment, or a differential abundance level of LCM at a single timepoint after treatment compared with normal levels of LCM, may beindicative of lung cancer progression, remission, or recurrence.

As a specific example, ELISA analysis of LCM levels in pre-surgery andpost-surgery (e.g., 2-4 weeks after surgery) serum samples can becarried out. An increase in the level of LCM in the post-surgery samplecompared with the pre-surgery sample can indicate progression of lungcancer (e.g., unsuccessful surgery), whereas a decrease in the level ofLCM in the post-surgery sample compared with the pre-surgery sample canindicate regression of lung cancer (e.g., the surgery successfullyremoved the lung tumor). Similar analyses of LCM levels can be carriedout before and after other forms of treatment, such as before and afterradiation therapy or administration of a therapeutic agent or cancervaccine.

In addition to the utilities of testing LCM levels as stand-alonescreening tests, testing of LCM levels can also be done in conjunctionwith CT screening. For example, LCM may facilitate the medical andeconomic justification for implementing CT screening, such as to screenlarge asymptomatic populations at risk for lung cancer (e.g., smokers).For example, a “pre-CT” test of LCM levels could be used to stratifyhigh-risk individuals for CT screening, such as to identify those whoare at highest risk for lung cancer based on their LCM levels and whoshould be prioritized for CT screening. If a CT test is implemented, LCMlevels (e.g., as determined by immunoassay of serum samples) of one ormore LCM can be measured and the scores added to scores for supplementalbiomedical parameters (e.g., tumor parameters determined by CT testing)to create a combined score, such as to enhance positive predictive value(PPV) over CT or LCM testing alone. A “post-CT” immunoassay panel fordetermining LCM levels can be used to determine the likelihood that apulmonary lesion observed by CT (or other imaging modality) is malignantor benign.

Detection of LCM may be useful for post-CT testing. For example, LCMtesting may eliminate a significant number of false positive tests overCT alone. Further, LCM testing may facilitate treatment of patients. Asan example, if a lung tumor is less than 5 mm in size, results of LCMtesting may move patients from “watch and wait” to biopsy at an earliertime, if a lung tumor is 5-9 mm, LCM testing may eliminate biopsy orthoracotomy on false positive scans, and if a lung tumor is larger than10 mm, LCM testing may eliminate surgery for sub-population of thesepatients with benign lesions Eliminating the need for biopsy in somepatients based on LCM testing would be beneficial because there issignificant morbidity associated with nodule biopsy and difficulty inobtaining nodule tissue depending on location of nodule. Similarly,eliminating the need for surgery in some patients, such as those whoselesions are actually benign, would avoid unnecessary risks and costsassociated with surgery.

In addition to testing LCM levels in conjunction with CT screening(e.g., assessing LCM levels in conjunction with size or othercharacteristics of a lung nodule observed on a CT scan), informationregarding LCM can also be evaluated in conjunction with other types ofdata, particularly data that indicates an individual's risk for lungcancer (e.g., patient clinical history, symptoms, family history ofcancer, risk factors such as whether or not the individual is a smoker,and/or status of other biomarkers, etc.). These various data can beassessed by automated methods, such as a computer program/software,which can be embodied in a computer or other apparatus/device.

The various methods described herein, such as correlating the level ofLCM in an individual with an altered (e.g., increased or decreased) risk(or no altered risk) for lung cancer, can be carried out by automatedmethods such as by using a computer (or other apparatus/devices such asbiomedical devices, laboratory instrumentation, or otherapparatus/devices having a computer processor) programmed to carry outany of the methods described herein. For example, computer software(which may be interchangeably referred to herein as a computer program)can perform the step of correlating the level of LCM in an individualwith an altered (e.g., increased or decreased) risk (or no altered risk)of lung cancer for the individual. Accordingly, certain embodiments ofthe invention provide a computer (or other apparatus/device) programmedto carry out any of the methods described herein.

LCM may also be used in imaging tests. For example, an imaging agent canbe coupled to an LCM, which can be used to aid in lung cancer diagnosis,to monitor disease progression/remission or metastasis, to monitor fordisease recurrence, or to monitor response to therapy, among other uses.

LCM can be detected using a variety of platforms. For example, LCM maybe detected using singleplex ELISAs, ultrasensitive detectiontechnologies, multiplex formats, and/or automated immunoanalyzers.

In addition to detecting LCM in serum, LCM may also be detected in, forexample, plasma and bronchial lavage.

LCM may be used for pharmacoproteomic or pharmacogenomic applications;for example, detection of LCM may be used for treatment selection orstratification. Differential expression of LCM in, for example, tumorcells that are resistant to a treatment (e.g., a particular therapeuticagent) and tumor cells that are sensitive to a treatment can be used topredict resistance or sensitivity of an individual's lung cancer to thetreatment. As specific examples, CTGF is secreted at elevated levels bycell lines that are resistant to the chemotherapeutic agent Topotecan.In contrast, TIMP1, TFPI, and TIMP2 are secreted at elevated levels bycell lines that are sensitive to the chemotherapeutic agent Iressa. LCMmay also be used as treatment response markers for a particulartherapeutic agent. For example, certain LCM may be used as surrogatemarkers of cisplatin or Iressa treatment response.

Thus, the LCM profile of an individual having lung cancer can be used todetermine which treatment(s) are best suited for that particularindividual. For example, treatments to which an individual's lung canceris predicted to be sensitive can be selected for the individual ratherthan treatments to which the individual's lung cancer is predicted to beresistant. As a further example, LCM levels can be used by a medicalpractitioner to distinguish between types of lung cancer (e.g.,non-small cell lung cancer (NSCLC) versus small cell lung cancer (SCLC),adenocarcinoma versus squamous cell carcinoma, different stages of lungcancer, or other lung cancer characteristics) in order to adjust therapyoptions (e.g., to select a particular therapeutic agent or a particularform of treatment, such as chemotherapy, surgery, or radiation therapy,that is best suited for that particular subtype of lung cancer).

Tables 5-6 and 11-12 provide panels that are particularly well-suitedfor diagnosing/detecting lung cancer, among other lung cancer-relateduses. For example, Tables 5 and 6 provides LCM panels that areparticularly well-suited for distinguishing lung tumor samples versusnormal (i.e., control/healthy) samples, Table 11 provides LCM panelsthat are particularly well-suited for distinguishing SCLC versus normalsamples, Table 12 provides LCM panels that are particularly well-suitedfor distinguishing lung cancer (including both SCLC and NSCLC) versusnormal samples.

Any of the LCM and the various exemplary LCM panels disclosed herein(such as any of the panels provided in Tables 5-6, such as the 9-markerpanel or the 6-marker subset of this 9-marker panel, as well as any LCMprovided in Tables 1-2 (SEQ ID NOS:1-65 and the carbohydrate antigens CA242, CA 19-9, and CA 72-4), Table 4, FIG. 1, and FIG. 12, and any panelsthat include one or more of these LCM, particularly panels that includeone or more markers provided in Table 4) may be used for any of thevarious lung cancer-related uses disclosed herein. However, certain LCMpanels are particularly well-suited for certain specific lungcancer-related uses (“indications”); these specific uses may be referredto herein as determining or assessing various “characteristics” of lungcancer. Examples of such LCM panels that are particularly well-suitedfor certain specific lung cancer-related uses are provided in Tables7-10 and FIG. 19. For example, Table 7 provides LCM panels that areparticularly well-suited for distinguishing adenocarcinoma versussquamous cell carcinoma types of lung cancer, Table 8 provides LCMpanels that are particularly well-suited for distinguishing betweendifferent stages of lung cancer (such as between early-stage andlate-stage lung cancer such as stage I versus stage III lung cancer, orbetween any other of stages I, II, III, and IV) such as to determine thestage of lung cancer in a patient, Table 9 provides LCM panels that areparticularly well-suited for distinguishing SCLC versus other types oflung cancer (e.g., NSCLC), Table 10 provides LCM panels that areparticularly well-suited for distinguishing malignant lung tumors versusbenign lung lesions, and FIG. 19 provides LCM that are particularlywell-suited for monitoring for lung tumor regression and/or recurrence.

Tables 5-12 provide a variety of exemplary LCM panels, together withperformance characterisitics for each panel (AUC, sensitivity, andspecificity). In certain embodiments, LCM panels are provided that haveat least 70% sensitivity at 95% specificity, or at least 70% specificityat 95% sensitivity. In certain embodiments, LCM panels are provided thathave at least 85% sensitivity at 95% specificity, or at least 85%specificity at 95% sensitivity. In further embodiments, LCM panels areprovided that have at least 90% sensitivity or at least 90% specificity,or that have at least 95% sensitivity or at least 95% specificity. Inyet further embodiments, LCM panels are provided that have at least 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% (or any otherpercentage in-between) sensitivity and 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% (or any other percentage in-between)specificity. In yet further embodiments, LCM panels are provided thathave at least 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95,0.96, 0.97, 0.98, or 0.99 (or any other value in-between) AUC values.Any of these panels are particularly useful for lung-cancer related usessuch as those described herein (e.g., diagnosing lung cancer), such asin clinical practice. However, the desired performance for clinical useof an assay may vary depending on such factors as the particular use,point of implementation, or other factors.

Distinguishing NSCLC and SCLC

The following panels of LCM are particularly useful for distinguishing(which may be interchangeably referred to as “resolving”) non-small celllung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) from eachother and/or from normal (i.e., control/healthy) samples. These panelsmay consist of, consist essentially of, or comprise the followingcombinations of markers:

-   -   1) Any of the panels provided in Table 9, particularly for        distinguishing NSCLC versus SCLC.    -   2) Any of the panels provided in Table 11, particularly for        distinguishing SCLC versus normal samples.    -   3) Any of the panels provided in Table 12, particularly for        distinguishing SCLC and NSCLC versus normal samples.    -   4) OPN (either alone or in combination with one or more other        markers), particularly for distinguishing NSCLC from SCLC and        for distinguishing SCLC from NSCLC.    -   5) SCC, OPN, AMBP, and Ca72-4, particularly for distinguishing        NSCLC from SCLC (levels of these LCM are higher in NSCLC as        compared to SCLC).    -   6) ENO2, MMP2, Ca19-9, CAIX, and GRP, particularly for        distinguishing SCLC from NSCLC (levels of these LCM are higher        in SCLC as compared to NSCLC).    -   7) Cyfra, SLPI, TIMP1, TFPI, CEACAM5, MMP2, and CA242,        particularly for distinguishing SCLC from normal samples        (particularly by using split-point analysis).    -   8) SLPI, TIMP1, TFPI, CEACAM5, MMP2, OPN, and CA242,        particularly for distinguishing SCLC from normal samples        (particularly by using split-point analysis).    -   9) Cyfra, TIMP1, ENO2, TFPI, CEACAM5, MMP2, OPN, and DEFA1,        particularly for distinguishing NSCLC and SCLC from normal        samples (particularly by using split-point analysis).    -   10) Cyfra, MIF, TIMP1, SCC, TFPI, CEACAM5, OPN, and DEFA1,        particularly for distinguishing NSCLC and SCLC from normal        samples (particularly by using split-point analysis).    -   11) TIMP1, ENO2, TFPI, CEACAM5, MMP2, OPN, AMBP, and DEFA1,        particularly for distinguishing NSCLC and SCLC from normal        samples (particularly by using split-point analysis).

Supplemental Biomedical Parameters

The term “supplemental biomedical parameters” refers to one or moreassessments of an individual, other than LCM, that are associated withlung cancer risk. “Supplemental biomedical parameters” include, but arenot limited to, physical descriptors of a patient, physical descriptorsof a pulmonary nodule observed by CT imaging, the height and/or weightof a patient, the gender of a patient, smoking history, occupationalhistory, exposure to carcinogens, exposure to second-hand smoke, familyhistory of lung cancer (or other cancer), the presence of pulmonarynodules, size of nodules, location of nodules, morphology of nodules(e.g., nodules may be observed by CT imaging), etc. Smoking history isusually quantified in terms of “pack years”, which refers to the numberof years a person has smoked multiplied by the average number of packssmoked per day. For example, a person who has smoked, on average, onepack of cigarettes per day for 35 years is referred to as having 35 packyears of smoking history. Supplemental biomedical parameters can beobtained from an individual using routine techniques known in the art,such as from the individual themselves by use of a routine patientquestionnaire or health history questionnaire, etc., or from a medicalpractitioner, etc. Alternately, supplemental biomedical parameters canbe obtained from routine imaging techniques including CT imaging (e.g.,low-dose CT imaging) and X-ray.

Testing of LCM in combination with an assessment of supplementalbiomedical parameters may, for example, improve sensitivity,specificity, and/or AUC for detecting lung cancer (or other lungcancer-related uses) as compared to LCM testing alone or assessingsupplemental biomedical parameters alone (e.g., CT imaging alone).

Accordingly, any of the LCM, and panels of LCM, can be used incombination with supplemental biomedical parameters. Furthermore,supplemental biomedical parameters may serve to replace one or moremarkers of a panel, such as to enable the use of smaller panels (i.e.,panels with fewer biomarkers) while retaining similar performance (e.g.,sensitivity, specificity, and/or AUC for detecting lung cancer). Thus,supplemental biomedical parameters can be used in addition to a panel,or in addition to one or more markers of a panel, or as a substitute forone or more markers of a panel. As a specific example, one or moresupplemental biomedical parameters can be used in addition to the9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, andMDK or the 6-marker subset of this 9-marker panel (Cyfra, SLPI, TIMP1,TFPI, CEACAM5, and MDK). As another specific example, one or moresupplemental biomedical parameters can replace one or more members ofthe 9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN,and MDK or the 6-marker subset of this 9-marker panel (Cyfra, SLPI,TIMP1, TFPI, CEACAM5, and MDK). Furthermore, one or more supplementalbiomedical parameters can be used in addition to any of the panelsprovided in Table 5 and/or can replace one or more members of any of thepanels provided in Table 5. Moreover, one or more supplementalbiomedical parameters can be used in addition to any of the markers orpanels provided herein and/or can replace one or more members of any ofthe panels provided herein, including the markers provided in Tables 1-2(SEQ ID NOS:1-65 and the carbohydrate antigens CA 242, CA 19-9, and CA72-4), Table 4, FIGS. 1, 12, and 19, and the panels provided in Tables 3and 6-12. Furthermore, supplemental biomedical parameters can beincorporated into algorithms and scoring systems/classifiers, togetherwith biomarker assessments (e.g., biomarker levels), for assessing lungcancer (e.g., diagnosing lung cancer).

Examples of supplemental biomedical parameters include, but are notlimited to, any of the following. Any or all of these supplementalbiomedical parameters can be used, in any combination, with any of theLCM and LCM panels disclosed herein. For example, any of the LCM and LCMpanels disclosed herein can be assessed alone (without consideringsupplemental biomedical parameters) or can be assessed in combinationwith CT results (for example), or can be assessed in combination withany other supplemental biomedical parameters, or can be assessed incombination with CT results plus any other supplemental biomedicalparameters, or any other combination of supplemental biomedicalparameters can be assessed in combination with any of the LCM and LCMpanels disclosed herein. Any of these supplemental biomedical parameterscan be assessed as part of an algorithm or scoring system/classifier,together with biomarker assessments (e.g., biomarker levels), such asfor assessing lung cancer (e.g., diagnosing lung cancer).

1) age, gender, and/or ethnicity;

2) family history of lung cancer or other type of cancer;

3) smoking history (e.g., whether or not an individual previously and/orcurrently smokes);

4) smoking level (e.g., “pack year”: number of cigarettes smoked per daymultiplied by number of years of smoking at this rate);

5) size of lesion;

6) location of lesion;

7) lesion morphology (ground glass opacity (GGO), solid, non-solid);

8) edge characteristics of lesion (smooth, lobulated, sharp and smooth,spiculated, infiltrating);

9) any other parameters determined from computed tomography (CT)screening;

10) exposure to second-hand smoke; and

10) any known carcinogen exposure (including, but not limited to,exposure to any of asbestos, radon gas, chemicals, smoke from fires, andair pollution, which can include emissions from stationary or mobilesources such as industrial/factory or auto/marine/aircraft emissions).

Exemplary methods of combining LCM with supplemental biomedicalparameters can comprise the steps of obtaining a value for at least onesupplemental biomedical parameter (e.g., smoking history) from anindividual, comparing the value of each of the supplemental biomedicalparameter(s) to one or more predetermined cutoffs, assigning a score foreach supplemental biomedical parameter based on said comparison,combining the assigned score for each supplemental biomedical parameterwith the assigned score for each LCM to obtain a total score for saidindividual, comparing the total score with a predetermined total scorecutoff, and classifying said individual as having or not having lungcancer (or the likelihood thereof) based on whether the individual'stotal score is above or below (or equal to) the predetermined totalscore cutoff. In certain embodiments, if the individual's total score isabove (or equal to) the predetermined total score cutoff, then theindividual is classified as having lung cancer.

Further exemplary methods can comprise the steps of:

a) obtaining a value for at least one supplemental biomedical parameterof an individual;

b) comparing the value of each supplemental biomedical parameter againstone or more predetermined cutoffs and assigning a score for eachsupplemental biomedical parameter based on said comparison;

c) quantifying in a test sample obtained from the individual, the levelsof one or more LCM or LCM panels (e.g., the 9-marker panel of Cyfra,SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK, or the 6-markersubset of Cyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK);

d) comparing the amount of each LCM quantified to a predetermined cutoffand assigning a score for each LCM based on said comparison;

e) combining the assigned score for each supplemental biomedicalparameter determined in step b with the assigned score for each LCMdetermined in step d to obtain a total score for said individual;

f) comparing the total score determined in step e with a predeterminedtotal score cutoff; and

g) classifying the individual (or the test sample from the individual)as having or not having lung cancer (or the likelihood thereof) based onwhether the individual's total score is above or below (or equal to) thepredetermined total score cutoff (in certain embodiments, if theindividual's total score is above (or equal to) the predetermined totalscore cutoff, then the individual is classified as having lung cancer).

In the above exemplary methods, the supplemental biomedical parameterobtained from the individual can be, for example, the individual'ssmoking history, age, carcinogen exposure, gender, nodule size, nodulemorphology, and/or nodule location (nodule characteristics, such assize, morphology, and/or location, may be determined by CT imaging, aswell as X-ray or other imaging methods). Preferably, the supplementalbiomedical parameter is related to nodule morphology.

Exemplary Scoring Systems and Cutoffs

A variety of methodologies can be used to classify a sample based onassaying one or more LCM disclosed herein. Classifying a sample can bebased on a score derived from assessing one or more LCM disclosedherein, optionally in combination with one or more supplementalbiomedical parameters (including, but not limited to, the supplementalbiomedical parameters disclosed in the preceding section). A score orother classification system can be based on, for example, determiningwhether the level of one or more LCM is above or below a cutoff level(which may be referred to as a “cutoff value” or just “cutoff”), or isabove or below a cutoff value by a certain amount (e.g., by a certainnumber of standard deviations such as two standard deviations), or themagnitude/extent of how high or low the level of one or more LCM is(which may optionally be in relation to a cutoff value). A wide varietyof scoring systems and methodologies for establishing cutoff values areknown in the art, and one of ordinary skill in the art would know how toimplement a known scoring system or method of establishing cutoff values(or devise a new scoring system or method for establishing cutoffvalues) that is best suited for the intended use, such as assessing lungcancer based on one or more LCM or LCM panels (optionally in combinationwith one or more supplemental biomedical parameters). Accordingly, oneof ordinary skill in the art could establish and adjust cutoff values tosuit the intended use, and could incorporate these cutoff values intoany desirable scoring system. For example, cutoff values can be adjustedbased on whether increased sensitivity (for detecting tumor samples andavoiding false-negatives) or increased specificity (for avoidingfalse-positives) is considered more important. For example, cutoffs canbe selected such as to achieve at least 70% sensitivity at 95%specificity, or at least 70% specificity at 95% sensitivity, or at least85% sensitivity at 95% specificity, or at least 85% specificity at 95%sensitivity, or at least 90% or 95% sensitivity, or at least 90% or 95%specificity, or any other desired sensitivity and/or specificity (suchas the sensitivity and specificity values described above). As anotherexample, cutoffs can be set lower while requiring more markers in apanel to be above the cutoff levels in order to classify a sample as atumor sample, or cutoffs can be set higher while requiring fewer markersin a panel to be above the cutoff levels in order to classify a sampleas a tumor sample. When a cutoff value is set and applied to testing, itmay be interchangeably referred to herein as a “predetermined” or“established” cutoff value. Furthermore, various analysis methods can beapplied, including, but not limited to, split-point analysis (such asfor setting discrete cutoffs), logistic regression analysis (such as forfactoring in the magnitude/extent by which a marker level is elevated orlow), Naïve Bayes, multivariate analysis, decision tree modeling, etc.

A representative example is shown in FIG. 17 for the 9-marker panel ofCyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK. In FIG. 17,exemplary cutoffs for each of these 9-markers are shown just below eachmarker symbol, as follows (levels/concentrations, in ng/ml, weredetermined by ELISA): Cyfra=1.20 ng/ml, CEA=5.00 ng/ml, SLPI=52 ng/ml,OPN=32 ng/ml, MDK=0.15 ng/ml, TFPI=150 ng/ml, TIMP1=385 ng/ml, MMP2=210ng/ml, and SCC=2.2 ng/ml. These cutoff values were established byexamining the levels of these markers in both normal (control) samplesand lung tumor samples in the 50×50 sample set and determiningappropriate cutoff values that would best distinguish lung tumor versusnormal samples (e.g., cutoff values were selected for which the levelsof a majority of lung tumor samples are above and the levels of amajority of normal samples are below, so as to maximize sensitivity andspecificity). These cutoffs were then applied to the same 50×50 sampleset (i.e., the 50×50 sample set was used for both training and testingin this example). In this example, if the levels of two or more of thenine markers was greater than or equal to the established cutoff valuefor each marker, then the sample was classified as a lung tumor sample(thus, if the levels of none, or only one, of the nine markers wasgreater than or equal to the established cutoff value for each marker,then the sample was classified as a normal sample). Using this exemplaryscoring system in this exemplary sample set, 48 out of 50 tumor sampleswere correctly classified, whereas the 42^(nd) and 43^(rd)-listedsamples were mis-classified as not being tumor samples since the levelof only one of the nine markers (rather than the minimum of two or more)in each of these two sample sets was greater than or equal to the cutofflevel (96% sensitivity; right-side of FIG. 17). Similarly, using thisexemplary scoring system in this exemplary sample set, 49 out of 50normal (control) samples were correctly classified, whereas the2^(nd)-listed sample was mis-classified as being a tumor sample sincethe levels of three of the nine markers (which meets the minimum of twoor more) in this sample set were greater than or equal to the cutofflevels (98% specificity; left-side of FIG. 17). However, one of skill inthe art would appreciate that no assay would be expected to correctlyclassify every single sample; rather, some misclassification is expectedin the art. The goal is generally to minimize, rather than eliminate,misclassifications. Further, an assay (such as an assay of LCM levels)can be combined with other types of tests (such as CT screening) tofurther minimize misclassifications.

In other exemplary scoring systems, if the levels of one or more, threeof more, four or more, five of more, six or more, seven or more, eightor more, or all nine markers of the 9-marker panel of Cyfra, SLPI,TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK are greater than or equalto the established cutoff value for each marker, then the sample can beclassified as a lung tumor sample. In certain exemplary scoring systems,if the level of a marker is greater than or equal to the establishedcutoff value for that marker, it can be assigned a value of one (forexample) and if the level of a marker is below the established cutoffvalue for that marker, it can be assigned a value of zero (for example).However, any desired values can be assigned to the various outcomes.Furthermore, these values can be added together (or otherwise combined)to determine a total score for a sample, and a classification of thesample as a tumor or normal sample (for example) can be assigned basedon this total score. For example, in the example described above anddepicted in FIG. 17, a score of two or greater can be used to classify asample as a tumor sample (and a score below two could be used toclassify a sample as a normal sample) if the level of each marker thatis greater than or equal to its established cutoff value is assigned avalue of one. Any other scoring system can be used, and one of ordinaryskill in the art would know how to select or devise a scoring systembest suited for the intended use.

A scoring system and cutoff values such as those exemplified in FIG. 17,or any other desirable scoring system and cutoff values, can be appliedto any of the other LCM and LCM panels disclosed herein, such as the6-marker subset of the 9-marker panel (Cyfra, SLPI, TIMP1, TFPI,CEACAM5, and MDK) and any of the other panels provided in Table 5, andcan optionally incorporate any supplemental biomedical parameters. Forexample, any supplemental biomedical parameters can be assigned a valuein a scoring system (e.g., a history of smoking can be assigned a valueof one or other value, and no smoking history can be assigned a value ofzero, negative one, or other value), and such values can be combinedwith the values assigned to marker levels being above a predeterminedcutoff level (for example), such as to generate a total score forclassifying a sample as a lung tumor or normal sample. Furthermore,these various scoring systems and cutoff values can be applied to any ofthe lung cancer-related uses disclosed herein, including specific usessuch as those disclosed in Tables 7-10.

Any of the scoring systems disclosed herein or known in the art, orwhich may be devised by one of ordinary skill in the art, can beincorporated into a computer program, and such a computer program can beembodied on computer readable medium. For example, a computer programcan generate a total score from a sample based on, for example, thenumber of markers in a panel for which the levels are abovepredetermined cutoff levels, together with parameters from CT screening(e.g., tumor volume/size, tumor morphology, tumor location, and/or othertumor characteristics, etc.) and/or other supplemental biomedicalparameters (e.g., smoking history, age of the individual, etc.), andthis total score can be used to classify a sample as a lung tumor ornormal sample, for example. A single total score can be generated thatrepresents the combination of multiple different types of assessments(e.g., a combination of LCM levels and supplemental biomedicalparameters), or multiple individual scores can be generated forevaluation individually (e.g., a score based on assessment of LCMlevels, and one or more separate scores based on supplemental biomedicalparameters). An example of this type of integrated approach of combiningLCM levels (using the exemplary panel of TIMP1, TFPI, CEACAM5, andCa72-4) with a supplemental biomedical parameter (smoking history, asindicated by “pack years”) is shown in FIG. 21.

Kits

Any combination of LCM and LCM panels (as well as supplementalbiomedical parameters) can be provided in the form of kits, such as foruse in performing the methods disclosed herein. Furthermore, any kit cancontain one or more detectable labels (e.g., detactably labeled reagentssuch as antibodies), such as a fluorescent moiety, etc.

For example, a kit can comprise (a) reagents comprising at least oneantibody for quantifying one or more LCM in a test sample, wherein saidLCM comprise: Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK(or just Cyfra, SLPI, TIMP1, TFPI, CEACAM5, and MDK; or any other LCM orLCM panels disclosed herein, such as the panels disclosed in Tables5-12), and optionally (b) one or more algorithms or computer programsfor performing the steps of comparing the amount of each LCM quantifiedin the test sample to one or more predetermined cutoffs and assigning ascore for each LCM quantified based on said comparison, combining theassigned score for each LCM quantified to obtain a total score,comparing the total score with a predetermined total score, and usingsaid comparison to determine whether an individual has lung cancer.Alternatively, rather than one or more algorithms or computer programs,one or more instructions for manually performing the above steps by ahuman can be provided.

In certain embodiments, a kit can contain: (a) reagents comprising atleast one antibody for quantifying one or more LCM in a test sample,wherein said LCM are Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN,and MDK, and (b) reagents containing one or more LCM for quantifying atleast one antibody in a test sample; wherein said antibodies are: TP53(p53), KLKB1, CFL1 (CFLN), EEF1G, HSP90α (HSP90AA1), RTN4, ALDOA, GLG1,PTK7, EFEMP1, SLC3A2 (CD98), CHGB, CEACAM1, ALCAM, HSPB1 (HSP27),LGALS1, and B7H3, and optionally (c) one or more algorithms or computerprograms for performing the steps of comparing the amount of each LCMand antibody quantified in the test sample to one or more predeterminedcutoffs and assigning a score for each LCM and antibody quantified basedon said comparison, combining the assigned score for each LCM andantibody quantified to obtain a total score, comparing the total scorewith a predetermined total score, and using said comparison to determinewhether an individual has lung cancer. Alternatively, rather than one ormore algorithms or computer programs, one or more instructions formanually performing the above steps by a human can be provided.

Translating LCM Assessments to Lung Cancer Assessments, and SystemsTherefor

An assessment of LCM in an individual, such as LCM levels determined byassaying a serum sample (or other sample) from the individual, can betranslated to an assessment of lung cancer for the individual. Forexample, the levels of multiple LCM (such as each of the LCM in the9-marker panel of Cyfra, SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, andMDK) can be translated to a score or other identifier that indicateswhether an individual has lung cancer (or that indicates the likelihoodthat the individual has lung cancer), for example. Similarly, the scoreor other identifier may indicate a specific type of lung cancerassessment, such as the assessments of various lung cancercharacteristics described herein, including (but not limited to),determination of whether an individual's lung cancer is adenocarcinomaor squamous cell carcinoma, determination of the stage of anindividual's lung cancer (such as distinguishing between stage I andstage III lung cancer), determination of whether an individual's lungcancer is SCLC or NSCLC, determining whether a lung lesion identified inan individual (such as by CT screening) is a malignant tumor or a benignlesion, and determining lung tumor regression and/or recurrence. Any ofthese determinations may be expressed in a discrete (e.g., absolute) orcontinuous (e.g., likelihood) manner, for example.

Furthermore, the assessment of LCM in an individual, such as LCM levels,can be translated to a tangible report. Thus, a score or otheridentifier that indicates the lung cancer assessment can be provided inthe form of a tangible report.

Additionally, the translation, such as the translation of LCM levels toa lung cancer assessment (such as a score or other identifier), can beperformed by a computer. Furthermore, certain embodiments providecomputer readable medium having a computer program code embodied thereonfor translating LCM levels to a lung cancer assessment.

In certain embodiments, the invention provides systems for assessing oneor more LCM, particularly the levels of multiple LCM, and translatingthis LCM assessment to an assessment of lung cancer, such as adetermination of whether an individual has (or is likely to have) lungcancer (which may be indicated by a score or other identifier). Incertain embodiments, these systems include one or more computers toreceive an LCM assessment, translate the LCM assessment to a lung cancerassessment, and output the lung cancer assessment (e.g., as a score orother identifier). These systems may optionally comprise multiplecomputers that communicate via the internet (or any other mode ofcommunication used in the art for inter-computer communication).

Accordingly, certain embodiments of the invention provide methods oftranslating an assessment of LCM (e.g., LCM levels) to an assessment oflung cancer (e.g., a score or other indication of whether an individualhas lung cancer, or their likelihood of having lung cancer, or otherspecific lung cancer assessment). In certain embodiments, thisassessment of lung cancer is provided in the form of a tangible report.In certain embodiments, the translation is performed by a computer.Furthermore, certain embodiments of the invention provide computersprogrammed to translate an LCM assessment to a lung cancer assessment.Certain embodiments provide computer readable medium having a computerprogram code embodied thereon for translating LCM levels to a lungcancer assessment. In certain embodiments, a system is provided forreceiving an LCM assessment, translating the LCM assessment to a lungcancer assessment, and outputting the lung cancer assessment (e.g., as ascore or other identifier). In various embodiments, the system comprisesone or more computers (which may optionally communicate via the internetor other mode of communication).

Reports, Transmission of Reports, and Programmed Computers

The results of a test (e.g., a diagnosis of lung cancer for anindividual based on the level, or other assay, of one or more LCMdisclosed herein, or assessment of tumorprogression/regression/recurrence, lung cancer stage, type of lungcancer such as NSCLC versus SCLC or adenocarcinoma versus squamous cellcarcinoma, malignant tumor versus benign lung lesion, etc.), and/or anyother information pertaining to a test (e.g., the levels of one or moreLCM disclosed herein in a sample from an individual, which mayoptionally be provided in the absence of explicit disease or diagnosticinformation), may be referred to herein as a “report”. A tangible reportcan optionally be generated as part of a testing process (which may beinterchangeably referred to herein as “reporting”, or as “providing” areport, “producing” a report, or “generating” a report).

Examples of tangible reports may include, but are not limited to,reports in paper (such as computer-generated printouts of test results)or equivalent formats and reports stored on computer readable medium(such as a CD, USB flash drive or other removable storage device,computer hard drive, or computer network server, etc.). Reports,particularly those stored on computer readable medium, can be part of adatabase, which may optionally be accessible via the internet (such as adatabase of patient records or biomedical information stored on acomputer network server, which may be a “secure database” that hassecurity features that limit access to the report, such as to allow onlythe patient and the patient's medical practitioners to view the reportwhile preventing other unauthorized individuals from viewing the report,for example). In addition to, or as an alternative to, generating atangible report, reports can also be displayed on a computer screen (orthe display of another electronic device or instrument).

A report can further be “transmitted” or “communicated” (these terms maybe used herein interchangeably), such as to the individual who wastested, a medical practitioner (e.g., a doctor, nurse, clinicallaboratory practitioner, etc.), a healthcare organization, a clinicallaboratory, and/or any other party or requester intended to view orpossess the report. The act of “transmitting” or “communicating” areport can be by any means known in the art, based on the format of thereport. Furthermore, “transmitting” or “communicating” a report caninclude delivering a report (“pushing”) and/or retrieving (“pulling”) areport. For example, reports can be transmitted/communicated by variousmeans, including being physically transferred between parties (such asfor reports in paper format) such as by being physically delivered fromone party to another, or by being transmitted electronically or insignal form (e.g., via e-mail or over the internet, by facsimile, and/orby any wired or wireless communication methods known in the art) such asby being retrieved from a database stored on a computer network server,etc.

In certain exemplary embodiments, the invention provides computers (orother apparatus/devices such as biomedical devices or laboratoryinstrumentation) programmed to carry out the methods described herein.For example, in certain embodiments, the invention provides a computerprogrammed to receive (i.e., as input) the levels of one or more LCMdisclosed herein and provide (i.e., as output) a lung cancer diagnosisor other result (e.g., assessment of tumorprogression/regression/recurrence, lung cancer stage, type of lungcancer such as NSCLC versus SCLC or adenocarcinoma versus squamous cellcarcinoma, malignant tumor versus benign lung lesion, etc.) based on thelevels of one or more LCM. Such output (e.g., communication of lungcancer diagnosis, etc.) may be, for example, in the form of a report oncomputer readable medium, printed in paper form, and/or displayed on acomputer screen or other display.

Further exemplary embodiments of the invention are described in greaterdetail below.

1. LCM Proteins

Exemplary embodiments of the invention provide LCM proteins that consistof, consist essentially of, or comprise the amino acid sequences of SEQID NOS:1-65 (additionally, carbohydrate antigens CA 242, CA 19-9, and CA72-4 are also provided, which may also be encompassed by referencesherein to proteins/polypeptides), as well as all known variants andfragments of these proteins, and nucleic acid molecules that are withinthe art to make and use. Examples of such obvious variants include, butare not limited to, naturally-occurring allelic variants, pre-processedor mature processed forms of a protein, non-naturally occurringrecombinantly-derived variants, orthologs, and paralogs. Such variantscan readily be generated using art-known techniques in the fields ofrecombinant nucleic acid technology and protein biochemistry.

A protein is said to be “isolated” or “purified” when it issubstantially free of cellular material or free of chemical precursorsor other chemicals. LCM proteins can be purified to homogeneity or otherdegrees of purity. The level of purification can be based on theintended use. The primary consideration is that the preparation allowsfor the desired function of the protein, even if in the presence ofconsiderable amounts of other components.

In some uses, “substantially free of cellular material” includespreparations of a protein having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the protein is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of a protein in which the protein isseparated from chemical precursors or other chemicals that are involvedin the protein's synthesis. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of a LCM protein having less than about 30% (by dry weight)chemical precursors or other chemicals, less than about 20% chemicalprecursors or other chemicals, less than about 10% chemical precursorsor other chemicals, or less than about 5% chemical precursors or otherchemicals.

Isolated LCM proteins can be purified from cells that naturally expressit, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods(e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd.ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001)). For example, a nucleic acid molecule encoding a LCM protein canbe cloned into an expression vector, the expression vector introducedinto a host cell, and the protein expressed in the host cell. Theprotein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques.

A LCM protein or fragment thereof can be attached to heterologoussequences to form chimeric or fusion proteins. Such chimeric and fusionproteins comprise a protein operatively linked to a heterologous proteinhaving an amino acid sequence not substantially homologous to theprotein. “Operatively linked” indicates that the protein and theheterologous protein are fused in-frame. The heterologous protein can befused to the N-terminus or C-terminus of the protein.

In some uses, the fusion protein does not affect the activity of theprotein per se. For example, the fusion protein can include, but is notlimited to, beta-galactosidase fusions, yeast two-hybrid GAL fusions,poly-His fusions, MYC-tagged, HI-tagged, and Ig fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant LCM proteins. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

A chimeric or fusion LCM protein can be produced by standard recombinantDNA techniques. For example, DNA fragments coding for different proteinsequences can be ligated together in-frame in accordance withconventional techniques. In another embodiment, a fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence(Ausubel et al., Current Protocols in Molecular Biology, 1992-2006).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST protein). A LCM-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the LCM protein.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences can be aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In an exemplary embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence can be alignedfor comparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions can then becompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein, amino acid or nucleic acid “identity” is equivalent toamino acid or nucleic acid “homology”). The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps, and the lengthof each gap, that are introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press,New York, 1991). In an exemplary embodiment, the percent identitybetween two amino acid sequences is determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package, usingeither a Blossom 62 matrix or a PAM250 matrix, a gap weight of 16, 14,12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. Inanother exemplary embodiment, the percent identity between twonucleotide sequences can be determined using the GAP program in the GCGsoftware package (Devereux et al., Nucleic Acids Res. 12(1):387 (1984))using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80,and a length weight of 1, 2, 3, 4, 5, or 6. In another exemplaryembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4.

The sequences of the proteins and nucleic acid molecules of theinvention can further be used as a “query sequence” to perform a searchagainst sequence databases to, for example, identify other proteinfamily members or related sequences. Such searches can be performedusing the NBLAST and XBLAST programs (version 2.0) of Altschul et al.(J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12, to obtainnucleotide sequences homologous to the query nucleic acid molecule.BLAST protein searches can be performed with the XBLAST program,score=50, wordlength=3, to obtain amino acid sequences homologous to thequery proteins. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

As used herein, two proteins (or a region or domain of the proteins)have significant homology/identity (also referred to as substantialhomology/identity) when the amino acid sequences are typically at leastabout 70-80%, 80-90%, 90-95%, 96%, 97%, 98%, or 99% identical Asignificantly homologous amino acid sequence can be encoded by a nucleicacid molecule that hybridizes to a LCM protein-encoding nucleic acidmolecule under stringent conditions, as more fully described below.

Orthologs of a LCM protein typically have some degree of significantsequence homology to at least a portion of a LCM protein and are encodedby a gene from another organism. Preferred orthologs are isolated frommammals, preferably non-human primates, for the development of humantherapeutic markers and agents. Such orthologs can be encoded by anucleic acid molecule that hybridizes to a LCM protein-encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

Non-naturally occurring variants of the LCM proteins can readily begenerated using recombinant techniques. Such variants include, but arenot limited to, deletions, additions, and substitutions in the aminoacid sequence of the LCM protein. For example, one class ofsubstitutions is conserved amino acid substitutions. Such substitutionsare those that substitute a given amino acid in a LCM protein by anotheramino acid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gln; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

Variant LCM proteins can be fully functional or can lack function in oneor more activities, e.g., ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variations orvariation in non-critical residues or in non-critical regions.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncations, or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity or in assays such as in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992); de Vos et al., Science 255:306-312(1992)).

LCM of the invention include fragments of LCM, and peptides thatcomprise and consist of such fragments. Such fragments of LCM may benaturally-occurring in the human body. An exemplary fragment typicallycomprises at least about 5, 6, 8, 10, 12, 14, 16, 18, 20 or morecontiguous amino acid residues of a LCM protein. Such fragments can bechosen based on the ability to retain one or more of the biologicalactivities of LCM or can be chosen for the ability to perform afunction, e.g., bind a substrate or act as an immunogen. Particularlyimportant fragments are biologically active fragments, such as peptidesthat are, for example, about 8 or more amino acids in length. Suchfragments can include a domain or motif of a LCM, e.g., an active site,a transmembrane domain, or a binding domain. Further, possible fragmentsinclude, but are not limited to, soluble peptide fragments and fragmentscontaining immunogenic structures. Domains and functional sites canreadily be identified, for example, by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).

Proteins can contain amino acids other than the 20 amino acids commonlyreferred to as the 20 naturally-occurring amino acids. Further, manyamino acids, including the terminal amino acids, can be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in proteins are wellknown to those of skill in the art.

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, tRNA-mediatedaddition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in the scientific literature. Several particularly commonmodifications, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, for instance, are described in most basic texts, suchas Proteins-Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this individual, such as by Wold(Posttranslational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press, New York 1-12 (1983)); Seifter et al. (Meth.Enzymol. 182: 626-646 (1990)); and Rattan et al. (Ann. N.Y. Acad. Sci.663:48-62 (1992)).

Accordingly, exemplary LCM proteins and fragments thereof of theinvention can also encompasses derivatives or analogs in which, forexample, a substituted amino acid residue is not one encoded by thegenetic code, in which a substituent group is included, in which amature LCM is fused with another composition, such as a composition toincrease the half-life of a LCM (e.g., polyethylene glycol or albumin),or in which additional amino acids are fused to a mature LCM, such as aleader or secretory sequence or a sequence for purification of a matureLCM or a pro-protein sequence.

2. Antibodies to LCM Proteins

Exemplary embodiments of the invention provide antibodies to LCMproteins, including, for example, monoclonal and polyclonal antibodies;chimeric, humanized, and fully human antibodies; and antigen-bindingfragments and variants thereof, as well as other embodiments.

Antibodies that selectively bind to a LCM protein can be made usingstandard procedures known to those of ordinary skills in the art. Theterm “antibody” is used in the broadest sense, and specifically covers,for example, monoclonal antibodies, polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), chimeric antibodies, humanizedantibodies, fully human antibodies, and antibody fragments (e.g., Fab,F(ab′)₂, Fv and Fv-containing binding proteins), so long as they exhibitLCM-binding activity. Antibodies (Ab's) and immunoglobulins (Ig's) areglycoproteins typically having the same structural characteristics.Antibodies can be of the IgG, IgE, IgM, IgD, and IgA class or subclassthereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Antibodies may beinterchangeably referred to as “LCM-binding molecules”.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population aresubstantially identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific and are typically directed against a singleantigenic site. Furthermore, in contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody istypically directed against a single determinant on an antigen. Inaddition to their specificity, monoclonal antibodies are advantageous inthat substantially homogenous antibodies can be produced by a hybridomaculture which is uncontaminated by other immunoglobulins or antibodies.The modifier “monoclonal” antibody indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, monoclonal antibodiescan be made by hybridoma methods such as described by Kohler andMilstein, Nature 256: 495-497 (1975), by recombinant methods (e.g., asdescribed in U.S. Pat. No. 4,816,567), or can be isolated from phageantibody libraries such as by using the techniques described in Clacksonet al., Nature 352: 624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597 (1991).

“Humanized” forms of non-human (e.g., murine or rabbit) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Typically, humanized antibodies are humanimmunoglobulins (a recipient antibody) in which residues from acomplementarity determining regions (“CDR”) of the recipient arereplaced by residues from a CDR of a non-human species (a donorantibody) such as mouse, rat, or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, a humanized antibody may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworkregion (FR) sequences. These modifications can be made to further refineand optimize antibody performance. In general, a humanized antibody cancomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theFRs are those of a human immunoglobulin consensus sequence. A humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details concerning humanized antibodies, see: Jones et al.,Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-327 (1988);Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Queen et al., U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370; and Winter,U.S. Pat. No. 5,225,539.

Antibodies, as used herein, include antibody fragments, particularlyantigen-binding fragments, as well as other modified antibody structuresand antigen-binding scaffolds (such as modified antibody structures thatare smaller or have less than all domains or chains compared with atypical naturally occurring, full-size human antibody). Examples ofantibody fragments and other modified antibody structures andantigen-binding scaffolds are known in the art by such terms asminibodies (e.g., U.S. Pat. No. 5,837,821), Nanobodies (llama heavychain antibodies; Ablynx, Ghent, Belgium), Adnectins (fibronectindomains; Adnexus Therapeutics, Waltham, Mass.), Affibodies(protein-binding domain of Staphylococcus aureus protein A; Affibody,Stockholm, Sweden), peptide aptamers (synthetic peptides; Aptanomics,Lyon, France), Avimers (A-domains derived from cell surface receptors;Avidia, Mountain View, Calif. (acquired by Amgen)), Transbodies(transferrin; BioRexis Pharmaceuticals, King of Prussia, Pa. (acquiredby Pfizer)), trimerized tetranectin domains (Borean Pharma, Aarhus,Denmark), Domain antibodies (heavy or light chain antibodies; Domantis,Cambridge, UK (acquired by GlaxoSmithKline)), Evibodies (derived fromV-like domains of T-cell receptors CTLA-4, CD28 and inducible T-cellcostimulator; EvoGenix Therapeutics, Sydney, Australia), scFV fragments(stable single chain antibody fragments; ESBATech, Zurich, Switzerland),Unibodies (monovalent IgG4 mAbs fragments; Genmab, Copenhagen, Denmark),BiTEs (bispecific, T-cell activating single-chain antibody fragments;Micromet, Munich, Germany), DARPins (designed ankyrin repeat proteins;Molecular Partners, Zurich, Switzerland), Anticalins (derived fromlipocalins; Pieris, Freising-Weihenstephan, Germany), Affilins (derivedfrom human lens protein gamma crystalline; Scil Proteins, Halle,Germany), and SMIPs (small modular immunopharmaceuticals; TrubionPharmaceuticals, Seattle, Wash.) (Sheridan, Nature Biotechnology, 2007April; 25(4):365-6).

An “isolated” or “purified” antibody is one that has been identified andseparated and/or recovered from a component of the environment in whichit is produced. Contaminant components of its production environment arematerials that would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In exemplary embodiments, the antibody canbe purified as measurable by any of at least three different methods: 1)to greater than 95% by weight of antibody as determined by the Lowrymethod, preferably more than 99% by weight; 2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator; or 3) to homogeneity bySDS-PAGE under reducing or non-reducing conditions using Coomasie blueor silver stain. Isolated antibody can include an antibody in situwithin recombinant cells since at least one component of the antibody'snatural environment will not be present. Ordinarily, however, anisolated antibody can be prepared by at least one purification step.

An “antigenic region”, “antigenic determinant”, or “epitope” includesany protein determinant capable of specific binding to an antibody. Thisis the site on an antigen to which each distinct antibody moleculebinds. Epitopic determinants can be active surface groupings ofmolecules such as amino acids or sugar side chains and may have specificthree-dimensional structural characteristics or charge characteristics.

“Antibody specificity” refers to an antibody that has a stronger bindingaffinity for an antigen from a first individual species than it has fora homologue of that antigen from a second individual species. Typically,an antibody “binds specifically” to a human antigen (e.g., has a bindingaffinity (Kd) value of no more than about 1×10⁻⁷ M, preferably no morethan about 1×10⁻⁸ M, and most preferably no more than about 1×10⁻⁹ M)but has a binding affinity for a homologue of the antigen from a secondindividual species which is at least about 50-fold, or at least about500-fold, or at least about 1000-fold, weaker than its binding affinityfor the human antigen. The antibodies can be of any of the various typesof antibodies as described herein, such as humanized or fully humanantibodies.

An antibody “selectively” or “specifically” binds a marker protein whenthe antibody binds the marker protein and does not significantly bind tounrelated proteins. An antibody can still be considered to selectivelyor specifically bind a marker protein even if it also binds to otherproteins that are not substantially homologous with the marker proteinas long as such proteins share homology with a fragment or domain of themarker protein. In this case, it would be understood that antibodybinding to the marker protein is still selective despite some degree ofcross-reactivity.

Exemplary embodiments of the invention provide an “antibody variant”,which refers to an amino acid sequence variant of an antibody whereinone or more of the amino acid residues have been modified. Such variantsnecessarily have less than 100% sequence identity with the amino acidsequence of the antibody, and have at least 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% amino acid sequence identity with the amino acidsequence of either the heavy or light chain variable domain of theantibody.

The term “antibody fragment” refers to a portion of a full-lengthantibody, including the antigen binding or variable region or theantigen-binding portion thereof. Examples of antibody fragments includeFab, Fab′, F(ab′)₂ and Fv fragments. Papain digestion of antibodiestypically produces two identical antigen binding fragments, called theFab fragment, each with a single antigen binding site, and a residual“Fc” fragment. Pepsin treatment typically yields an F(ab′)₂ fragmentthat has two antigen binding fragments which are capable of crosslinkingantigen, and a residual other fragment (which is termed pFc′). Examplesof additional antigen-binding fragments can include diabodies,triabodies, tetrabodies, single-chain Fv, single-chain Fv-Fc, SMIPs, andmultispecific antibodies formed from antibody fragments. A “functionalfragment”, with respect to antibodies, typically refers to an Fv, F(ab),F(ab′)₂ or other antigen-binding fragments comprising one or more CDRsthat has substantially the same antigen-binding specificity as anantibody.

An “Fv” fragment is an example of an antibody fragment that contains acomplete antigen recognition and binding site. This region typicallyconsists of a dimer of one heavy and one light chain variable domain ina tight, non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen.

An “Fab” fragment (also designated as “F(ab)”) also contains theconstant domain of the light chain and the first constant domain (CH1)of the heavy chain. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxyl terminus of the heavy chainCH1 domain, including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation for Fab′ in which the cysteineresidue(s) of the constant domains have a free thiol group. F(ab′)fragments are produced by cleavage of the disulfide bond at the hingecysteines of the F(ab′)₂ pepsin digestion product. Additional chemicalcouplings of antibody fragments are known to those of ordinary skill inthe art.

A “single-chain Fv” or “scFv” antibody fragment contains V_(H) and V_(L)domains, wherein these domains are present in a single polypeptidechain. Typically, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains that enables the scFv to formthe desired structure for antigen binding. For a review of scFv, seePlückthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).A single chain Fv-Fc is an scFv linked to a Fc region.

A “diabody” is a small antibody fragment with two antigen-binding sites,which fragments comprise a variable heavy domain (V_(H)) connected to avariable light domain (V_(L)) in the same polypeptide chain. By using alinker that is too short to allow pairing between the two domains on thesame chain, the domains are forced to pair with the complementarydomains of another chain and create two antigen-binding sites. Diabodiesare described more fully in, for example, EP 0 404 097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).Triabodies, tetrabodies and other antigen-binding antibody fragmentshave been described by Hollinger and Hudson, 2005, Nature Biotechnology23:1126.

A “small modular immunopharmaceutical” (or “SMIP”) is a single-chainpolypeptide including a binding domain (e.g., an scFv or an antigenbinding portion of an antibody), a hinge region, and an effector domain(e.g., an antibody Fc region or a portion thereof). SMIPs are describedin published U.S. Patent Application No. 20050238646.

Many methods are known for generating and/or identifying antibodies to agiven marker protein. Several such methods are described by Kohler etal., 1975, Nature 256: 495-497; Lane, 1985, J. Immunol. Meth.81:223-228; Harlow et al., 1988, Antibodies: A Laboratory Manual. ColdSpring Harbor Laboratory Press; Harlow et al., 1998, Using Antibodies,Cold Spring Harbor Press; Zhong et al., 1997, J. Indust. Microbiol.Biotech. 19(1):71-76; and Berry et al., 2003, Hybridoma and Hybridomics22(1): 23-31.

Polyclonal antibodies can be prepared by any known method ormodifications of these methods, including obtaining antibodies frompatients. In certain exemplary methods for generating antibodies such aspolyclonal antibodies, an isolated protein can be used as an immunogenwhich is administered to a mammalian organism, such as a rat, rabbit, ormouse. For example, a complex of an immunogen such as a LCM protein (orfragment thereof) and a carrier protein can be prepared and an animalimmunized by the complex. Serum or plasma containing antibodies againstthe protein can be recovered from the immunized animal and theantibodies separated and purified (in the same manner as for monoclonalantibodies, for example). The gamma globulin fraction or the IgGantibodies can be obtained, for example, by use of saturated ammoniumsulfate or DEAE SEPHADEX, or other techniques known to those skilled inthe art. The antibody titer in the antiserum can be measured in the samemanner as in the supernatant of a hybridoma culture.

A marker such as a full-length LCM protein, an antigenic peptidefragment, a fusion protein thereof, or a carbohydrate antigen orfragment thereof, can be used as an immunogen. A marker used as animmunogen is not limited to any particular type of immunogen. In oneaspect, antibodies can be prepared from regions or discrete fragments(e.g., functional domains, extracellular domains, or portions thereof)of a LCM. Antibodies can be prepared from any region of a marker asdescribed herein. In particular, the markers can be selected from thegroup consisting of SEQ ID NOS:1-65, the carbohydrate antigens CA 242,CA 19-9, and CA 72-4, and fragments thereof. An antigenic fragment cantypically comprise at least 8, 10, 12, 14, 16, or more contiguous aminoacid residues, for example. Such fragments can be selected based on aphysical property, such as fragments that correspond to regions locatedon the surface of a marker (e.g., hydrophilic regions) or can beselected based on sequence uniqueness.

Antibodies can also be produced by inducing production in a lymphocytepopulation or by screening antibody libraries or panels of highlyspecific binding reagents, such as disclosed in Orlandi et al. (Proc.Natl. Acad. Sci. 86:3833-3837 (1989)) or Winter et al. (Nature349:293-299 (1991)). A protein can be used in screening assays ofphagemid or B-lymphocyte immunoglobulin libraries to identify antibodieshaving a desired specificity. Numerous protocols for competitive bindingor immunoassays using either polyclonal or monoclonal antibodies withestablished specificities are well known in the art (e.g., Smith, Curr.Opin. Biotechnol. 2: 668-673 (1991)).

Antibodies can also be generated using various phage display methodsknown in the art. In representative phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry nucleic acid molecules that encode the antibody domains. Inparticular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds an antigen of interest can be selected or identified with theantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in methods such as these can typicallybe filamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv, or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make antibodiesinclude methods described in Brinkman et al., J. Immunol. Methods182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187:9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

Antibodies, antigen binding fragments, and/or antibody variants can beproduced by recombinant and genetic engineering methods well known inthe art. For example, methods of expressing heavy and light chain genesin E. coli are described in PCT publication numbers WO901443, WO901443,and WO9014424, and in Huse et al., 1989 Science 246:1275-1281. Whenusing recombinant techniques, such as to produce an antibody variant,the antibody variant can be produced intracellularly, in the periplasmicspace, or directly secreted into the medium. If an antibody variant isproduced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, can be removed, for example, bycentrifugation or ultrafiltration. Carter et al. (Bio/Technology 10:163-167 (1992)) describe a procedure for isolating antibodies that aresecreted to the periplasmic space of E. coli. Briefly, cell paste can bethawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where an antibody variant is secretedinto the medium, supernatants from such expression systems can first beconcentrated using a commercially available protein concentration filter(e.g., an Amicon or Millipore PELLICON ultrafiltration unit). A proteaseinhibitor such as PMSF can be included in any of the foregoing steps toinhibit proteolysis, and antibiotics can be included to prevent thegrowth of contaminating microorganisms.

An antibody composition prepared from cells can be purified using, forexample, affinity chromatography, hydroxylapatite chromatography, gelelectrophoresis, and/or dialysis. The suitability of protein A as anaffinity ligand typically depends on the species and isotype of theimmunoglobulin Fc domain of an antibody. Protein A can be used to purifyantibodies that are based on human delta1, delta2, or delta4 heavychains (Lindmark et al., J. Immunol Meth. 62: 1-13 (1983)). Protein Gcan be used for all mouse isotypes and for human delta3 (Guss et al.,EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity ligand isattached can be, for example, agarose or mechanically stable matricessuch as controlled pore glass or poly(styrenedivinyl)benzene. Where theantibody comprises a CH3 domain, the BAKERBOND ABX™ resin (J. T. Baker,Phillipsburg, N.J.) can be used for purification. Other exemplarytechniques for antibody purification include, but are not limited to,fractionation on an ion-exchange column, ethanol precipitation, reversephase HPLC, chromatography on silica, chromatography on heparinhepharos, chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation.

Following any preliminary purification step(s), contaminants in amixture containing an antibody of interest can be removed by low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Full-length antibodies, as well as antibody fragments, can also beexpressed and isolated from bacteria such as E. coli, such as describedin Mazor et al., “Isolation of engineered, full-length antibodies fromlibraries expressed in Escherichia coli”, Nat Biotechnol. 2007 May;25(5):563-5 and Sidhu, “Full-length antibodies on display”, NatBiotechnol. 2007 May; 25(5):537-8.

Further details regarding antibodies are set forth in the following U.S.Pat. No. 6,248,516 (Winter et al.); U.S. Pat. No. 6,291,158 (Winter etal.); U.S. Pat. No. 5,885,793 (Griffiths et al.); U.S. Pat. No.5,969,108 (McCafferty et al.); U.S. Pat. No. 5,939,598 (Kucherlapati etal.); U.S. Pat. No. 4,816,397 (Boss et al.); U.S. Pat. No. 4,816,567(Cabilly et al.); U.S. Pat. No. 6,331,415 (Cabilly et al.); U.S. Pat.No. 5,770,429 (Lonberg et al.); U.S. Pat. No. 5,639,947 (Hiatt et al.);and U.S. Pat. No. 5,260,203 (Ladner et al.), each of which isincorporated herein by reference, and in the following published U.S.patent applications: US20040132101 (Lazar et al.), US20050064514(Stavenhagen et al.), US20040261148 (Dickey et al.), and US20050014934(Hinton et al.), each of which is incorporated herein by reference.Antibody engineering is further described in Jain et al., “Engineeringantibodies for clinical applications”, Trends Biotechnol. 2007 July;25(7):307-16.

3. Antibody-Drug Conjugates to LCM Proteins

An antibody against LCM can be coupled (e.g., covalently bonded) to asuitable therapeutic agent (as further discussed herein) either directlyor indirectly (e.g., via a linker group). A direct reaction between anantibody and a therapeutic agent is possible when each possesses asubstituent capable of reacting with the other. For example, anucleophilic group, such as an amino or sulfhydryl group, on onemolecule may be capable of reacting with a carbonyl-containing group,such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other molecule.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

A variety of bifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), can be employed as the linker group.Coupling can be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups, or oxidized carbohydrate residues (e.g., U.S.Pat. No. 4,671,958).

Where a therapeutic agent is more potent when free from the antibodyportion of an immunoconjugate, it may be desirable to use a linker groupthat is cleavable during or upon internalization into a cell. A numberof different cleavable linker groups have been described. Mechanisms forthe intracellular release of an agent from these linker groups includecleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.4,489,710), by irradiation of a photolabile bond (e.g., U.S. Pat. No.4,625,014), by hydrolysis of derivatized amino acid side chains (e.g.,U.S. Pat. No. 4,638,045), by serum complement-mediated hydrolysis (e.g.,U.S. Pat. No. 4,671,958), by protease cleavable linker (e.g., U.S. Pat.No. 6,214,345), and by acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789).

It may be desirable to couple more than one agent to an antibody.Multiple molecules of an agent can be coupled to one antibody molecule,and more than one type of agent can be coupled to the same antibody. Forexample, about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 (or anyother number in-between) molecules of therapeutic agents can be coupledto an antibody. The average number or quantitative distribution oftherapeutic agent molecules per antibody molecule in a preparation ofconjugation reactions can be determined by conventional means such asmass spectroscopy, ELISA, or HPLC. Separation, purification, andcharacterization of homogeneous antibody-drug conjugates having acertain number of therapeutic agents conjugated thereto can be achievedby means such as reverse phase HPLC or electrophoresis (see, e.g.,Hamblett et al., Clinical Cancer Res. 10:7063-70 (2004).

Examples of suitable therapeutic agents that can be conjugated to anantibody include, but are not limited to, chemotherapeutic agents (e.g.,cytotoxic or cytostatic agents or immunomodulatory agents),radiotherapeutic agents, therapeutic antibodies, small molecule drugs,peptide drugs, immunomodulatory agents, differentiation inducers, andtoxins.

Examples of useful classes of cytotoxic or immunomodulatory agentsinclude, but are not limited to, antitubulin agents, auristatins, DNAminor groove binders, DNA replication inhibitors, alkylating agents(e.g., platinum complexes such as cis-platin, mono(platinum),bis(platinum) and tri-nuclear platinum complexes and carboplatin),anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapysensitizers, duocarmycins, etoposides, fluorinated pyrimidines,ionophores, lexitropsins, nitrosoureas, platinols, pre-formingcompounds, purine antimetabolites, puromycins, radiation sensitizers,steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and thelike.

Examples of individual cytotoxic or immunomodulatory agents include, butare not limited to, androgen, anthramycin (AMC), asparaginase,5-azacytidine, azathioprine, bleomycin, busulfan, buthioninesulfoximine, calicheamicin or calicheamicin derivatives, camptothecin orcamptothecins derivatives, carboplatin, carmustine (BSNU), CC-1065,chlorambucil, cisplatin, colchicine, cyclophosphamide, cytidinearabinoside (cytarabine), cytochalasin B, dacarbazine, dactinomycin(formerly actinomycin), daunorubicin, decarbazine, docetaxel,doxorubicin, etoposide, estrogen, 5-fluordeoxyuridine, 5-fluorouracil,gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine,rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA,topotecan, vinblastine, vincristine, vinorelbine, VP-16, and VM-26.

Examples of other suitable cytotoxic agents include, but are not limitedto, DNA minor groove binders (e.g., enediynes and lexitropsins, a CBIcompound; see also U.S. Pat. No. 6,130,237), duocarmycins, taxanes(e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065,SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin,epothilone A and B, estramustine, cryptophysins, cemadotin, amaytansinoid, discodermolide, eleutherobin, and mitoxantrone.

Examples of other suitable agents include, but are not limited to,radionuclides, differentiation inducers, drugs, toxins, and derivativesthereof. Exemplary radionuclides ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re,²¹¹At, and ²¹²Bi. Exemplary drugs include methotrexate, and pyrimidineand purine analogs. Exemplary differentiation inducers include phorbolesters and butyric acid. Exemplary toxins include ricin, abrin,diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigellatoxin, and pokeweed antiviral protein.

In some embodiments, the therapeutic agent used in an antibody-drugconjugate is an anti-tubulin agent. Examples of anti-tubulin agentsinclude, but are not limited to, taxanes (e.g., Taxol® (paclitaxel),Taxotere® (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g.,vincristine, vinblastine, vindesine, and vinorelbine). Other antitubulinagents include, for example, baccatin derivatives, taxane analogs (e.g.,epothilone A and B), nocodazole, colchicine and colcimid, estramustine,cryptophysins, cemadotin, maytansinoid, combretastatins, discodermolide,and eleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine, DM-1 (ImmunoGen, Inc.; see also Chari etal., Cancer Res. 52:127-131 (1992)) or DM-4. In some embodiments, thetherapeutic agent is an auristatin, such as auristatin E (also known inthe art as dolastatin-10) or a derivative thereof. Typically, anauristatin E derivative is, e.g., an ester formed between auristatin Eand a keto acid. For example, auristatin E can be reacted withparaacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB,respectively. Other typical auristatin derivatives include AFP, MMAF,and MMAE. The synthesis and structure of auristatin derivatives aredescribed in U.S. Patent Application Publication Nos. 2003-0083263,2005-0238649 and 2005-0009751; PCT Publication Nos WO 04/010957 and WO02/088172, and U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065;5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725;5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973;4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.

4. LCM Nucleic Acid Molecules

Exemplary isolated LCM nucleic acid molecules of the invention consistof, consist essentially of, or comprise a nucleotide sequence thatencodes a LCM protein of the invention, an allelic variant thereof, oran ortholog or paralog thereof, for example. As used herein, an“isolated” nucleic acid molecule is one that is separated from othernucleic acid present in the natural source of the nucleic acid.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. However, there can be some flankingnucleotide sequences, for example up to about 5 kilobases (KB), 4 KB, 3KB, 2 KB, or 1 KB or less, particularly contiguous protein-encodingsequences and protein-encoding sequences within the same gene butseparated by introns in the genomic sequence, and flanking nucleotidesequences that contain regulatory elements. The primary consideration isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be individualed to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid molecules.Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated. Isolated nucleic acidmolecules can include heterologous nucleotide sequences, such asheterologous nucleotide sequences that are fused to a nucleic acidmolecule by recombinant techniques. For example, recombinant DNAmolecules contained in a vector are considered isolated. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells, or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of isolated DNA molecules. Isolatednucleic acid molecules further include such molecules producedsynthetically.

Isolated nucleic acid molecules can encode a mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature protein (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life, or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, additional amino acids may beprocessed away from the mature protein by cellular enzymes.

Isolated nucleic acid molecules include, but are not limited to,sequences encoding a LCM protein alone, sequences encoding a matureprotein with additional coding sequences (such as a leader or secretorysequence (e.g., a pre-pro or pro-protein sequence)), and sequencesencoding a mature protein (with or without additional coding sequences)plus additional non-coding sequences (e.g., introns and non-coding 5′and 3′ sequences such as transcribed but non-translated sequences thatplay a role in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding, and/or stability of mRNA).In addition, nucleic acid molecules can be fused to a marker sequenceencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form of DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. Nucleic acid molecules, especially DNA, can be double-strandedor single-stranded. Single-stranded nucleic acid can be the codingstrand (sense strand) or the non-coding strand (anti-sense strand).

Exemplary embodiments of the invention further provide isolated nucleicacid molecules that encode fragments of a LCM protein as well as nucleicacid molecules that encode obvious variants of a LCM protein. Suchnucleic acid molecules may be naturally occurring, such as allelicvariants (same locus), paralogs (different locus), and orthologs(different organism), or can be constructed by recombinant DNA methodsor by chemical synthesis. Such non-naturally occurring variants can bemade by mutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, nucleic acid moleculevariants can contain nucleotide substitutions, deletions, inversions,and/or insertions. Variations can occur in either or both the coding andnon-coding regions, and variations can produce conservative and/ornon-conservative amino acid substitutions.

A fragment of a nucleic acid molecule typically comprises a contiguousnucleotide sequence at least 8, 10, 12, 15, 16, 18, 20, 22, 25, 30, 40,50, 100, 150, 200, 250, 500 (or any other number in-between) or morenucleotides in length. The length of a fragment can be based on itsintended use. For example, a fragment can encode epitope bearing regionsof a protein, or can be used as DNA probes and primers. Isolatedfragments can be produced by synthesizing an oligonucleotide probe usingknown techniques, for example, and can optionally be labeled and used toscreen a cDNA library, genomic DNA, or mRNA, for example. Primers can beused in PCR reactions to clone specific regions of a gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. An oligonucleotide typicallycomprises a nucleotide sequence that hybridizes under stringentconditions to at least about 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 40, 50 (or any other number in-between) or morecontiguous nucleotides.

Allelic variants, orthologs, and homologs can be identified usingmethods well known in the art. These variants can comprise a nucleotidesequence encoding a protein that is typically 60-70%, 70-80%, 80-90%,90-95%, 96%, 97%, 98%, or 99% homologous to the nucleotide sequence.Such nucleic acid molecules can readily be identified as being able tohybridize under moderate to stringent conditions, to a nucleotidesequence shown in the Sequence Listing or a fragment thereof.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a protein at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in, for example, Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989-2006), 6.3.1-6.3.6.One example of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50-65° C. Examples of moderate tolow stringency hybridization conditions are well known in the art.

Exemplary embodiments of the invention also include kits for detectingthe presence of LCM nucleic acid (e.g., DNA or mRNA) in a biologicalsample. For example, a kit can comprise reagents such as a labeled orlabelable nucleic acid and/or other agents capable of detecting LCMnucleic acid in a biological sample; means for determining the amount ofLCM nucleic acid in the sample; and means for comparing the amount ofLCM nucleic acid in the sample with a standard. The nucleic acid and/orother agent can be packaged in one or more suitable containers. The kitcan further comprise instructions for using the kit to detect LCMnucleic acid.

5. Vectors and Host Cells

Exemplary embodiments of the invention also provide vectors containingLCM nucleic acid molecules. The term “vector” refers to a vehicle, suchas a nucleic acid molecule, which can transport the LCM nucleic acidmolecules. When the vector is a nucleic acid molecule, the LCM nucleicacid molecules are covalently linked to the vector nucleic acid. Avector can be, for example, a plasmid, single or double stranded phage,a single or double stranded RNA or DNA viral vector, or artificialchromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in a host cell as an extrachromosomal elementwhere it replicates and produces additional copies of the LCM nucleicacid molecules. Alternatively, a vector can integrate into a host cellgenome and produce additional copies of the LCM nucleic acid moleculeswhen the host cell replicates.

Exemplary embodiments of the invention provide vectors for maintenance(cloning vectors) and vectors for expression (expression vectors) of thenucleic acid molecules, for example. Expression vectors can express aportion of, or all of, a protein sequence. Vectors can function inprokaryotic or eukaryotic cells or in both (shuttle vectors). Vectorsalso include insertion vectors, which integrate a nucleic acid moleculeinto another nucleic acid molecule, such as into the cellular genome(such as to alter in situ expression of a gene and/or gene product). Forexample, an endogenous protein-coding sequence can be entirely orpartially replaced via homologous recombination with a protein-codingsequence containing one or more specifically introduced mutations.

Expression vectors can contain cis-acting regulatory regions that areoperably-linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Theseparate nucleic acid molecule may provide, for example, a trans-actingfactor interacting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by a host cell.Additionally, a trans-acting factor can be produced from a vectoritself. It is understood, however, that transcription and/or translationof nucleic acid molecules can occur in cell-free systems.

Regulatory sequences to which LCM nucleic acid molecules can be operablylinked include, for example, promoters for directing mRNA transcription.These include, but are not limited to, the left promoter frombacteriophage, the lac, TRP, and TAC promoters from E. coli, the earlyand late promoters from SV40, the CMV immediate early promoter, theadenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors can also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region, a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. Numerous regulatory sequences useful inexpression vectors are well known in the art (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001)).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001).

A regulatory sequence can provide constitutive expression in one or morehost cells (e.g., tissue specific) or can provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factors such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known in the art.

Nucleic acid molecules can be inserted into vector nucleic acid bywell-known methodology. For example, the DNA sequence that willultimately be expressed can be joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known in the art.

A vector containing a nucleic acid molecule of interest can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells (e.g., DG44or CHO-s), and plant cells.

As described herein, it may be desirable to express a protein as afusion protein. Accordingly, exemplary embodiments of the inventionprovide fusion vectors that allow for the production of fusion proteins.Fusion vectors can, for example, increase the expression of arecombinant protein; increase the solubility of a recombinant protein,and/or aid in the purification of a protein such as by acting as aligand for affinity purification. A proteolytic cleavage site can beintroduced at the junction of the fusion moiety so that the desiredprotein can ultimately be separated from the fusion moiety. Proteolyticenzymes include, but are not limited to, factor Xa, thrombin, andenteroenzyme. Typical fusion expression vectors include pGEX (Smith etal., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.)and pRIT5 (Pharmacia, Piscataway, N.J.), which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to a recombinant marker protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990), pp. 119-128). Alternatively, thesequence of a nucleic acid molecule of interest can be altered toprovide preferential codon usage for a specific host cell, such as E.coli (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

LCM nucleic acid molecules can, for example, be expressed by expressionvectors in a yeast host. Examples of vectors for expression in yeast(e.g., S. cerevisiae) include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943 (1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.). Nucleic acid molecules can also beexpressed in insect cells using, for example, baculovirus expressionvectors. Baculovirus vectors available for expression of proteins incultured insect cells (e.g., Sf 9 cells) include the pAc series (Smithet al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklowet al., Virology 170:31-39 (1989)). Nucleic acid molecules can also beexpressed in mammalian cells using mammalian expression vectors.Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature329:840 (1987)), pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)), andCHEF (U.S. Pat. No. 5,888,809).

The expression vectors listed herein are provided by way of example onlyof well-known vectors available to those of ordinary skill in the artthat would be useful to express LCM nucleic acid molecules. The personof ordinary skill in the art would be aware of other vectors suitablefor maintenance, propagation, and/or expression of LCM nucleic acidmolecules (e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual.3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).

Exemplary embodiments of the invention also encompasses vectors in whichLCM nucleic acid molecules are cloned into a vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of a LCM nucleic acid molecule,including coding and non-coding regions. Expression of this antisenseRNA may be individual to each of the parameters described above inrelation to expression of the sense RNA (e.g., regulatory sequences,constitutive or inducible expression, tissue-specific expression).

Exemplary embodiments of the invention provide recombinant host cellscontaining the vectors described herein. Host cells include, forexample, prokaryotic cells, lower eukaryotic cells such as yeast, othereukaryotic cells such as insect cells, and higher eukaryotic cells suchas mammalian cells.

Recombinant host cells can be prepared by introducing vector constructs,such as described herein, into cells by techniques readily available toa person of ordinary skill in the art. These techniques include, but arenot limited to, calcium phosphate transfection, DEAE-dextran-mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, lipofection, microinjection, and othertechniques such as those found in Sambrook, et al. (Molecular Cloning: ALaboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001).

For example, using techniques such as these, a retroviral or other viralvector can be introduced into mammalian cells. Examples of mammaliancells into which a retroviral vector can be introduced include, but arenot limited to, primary mammalian cultures or continuous mammaliancultures, COS cells, NIH3T3, 293 cells (ATCC #CRL 1573), and dendriticcells.

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, nucleic acid molecules of interest can be introduced eitheralone or with other unrelated nucleic acid molecules such as thoseproviding trans-acting factors for expression vectors. When more thanone vector is introduced into a cell, the vectors can be introducedindependently, co-introduced, or joined to the nucleic acid moleculevector.

Bacteriophage and viral vectors can be introduced into cells as packagedor encapsulated virus by standard procedures for infection andtransduction. Viral vectors can be replication-competent orreplication-defective. If viral replication is defective, replicationcan occur in host cells that provide functions that complement thedefects.

Vectors can include selectable markers that enable the selection of asubpopulation of cells that contain the recombinant vector constructs.Markers can be contained in the same vector that contains the nucleicacid molecules of interest or can be on a separate vector. Exemplarymarkers include tetracycline or ampicillin-resistance genes forprokaryotic host cells, and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait can be used.

While mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

If secretion of a protein is desired, appropriate secretion signals canbe incorporated into a vector. The signal sequence can be endogenous orheterologous to the protein.

If a protein is not secreted into a medium, the protein can be isolatedfrom a host cell by standard disruption procedures, includingfreeze/thaw, sonication, mechanical disruption, use of lysing agents,and the like. A protein can then be recovered and purified by well-knownpurification methods including, for example, ammonium sulfateprecipitation, acid extraction, anion or cationic exchangechromatography, phosphocellulose chromatography, hydrophobic-interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, or high performance liquid chromatography.

It is also understood that, depending upon the host cell used inrecombinant production of a protein, proteins can have variousglycosylation patterns or can be non-glycosylated, such as when producedin bacteria. In addition, proteins can include an initial modifiedmethionine in some instances as a result of a host-mediated process.

Recombinant host cells that express a LCM protein have a variety ofuses. For example, such host cells are useful for producing LCMproteins, which can be further purified to produce desired amounts ofthe protein or fragments thereof. Thus, host cells containing expressionvectors are useful for protein production.

Host cells are also useful for conducting cell-based assays involving aLCM protein or fragments thereof. For example, a recombinant host cellexpressing a LCM protein can be used to assay compounds that stimulateor inhibit the protein's function.

Host cells are also useful for identifying mutant LCM proteins in whichthe protein's function is affected. Host cells expressing mutantproteins are useful for assaying compounds that have a desired effect onthe mutant proteins (e.g., stimulating or inhibiting function),particularly if the mutant proteins naturally occur and give rise to apathology.

6. Diagnosis and Treatment in General

The following terms, as used in the present specification and claims,are intended to have the meaning as defined below, unless indicatedotherwise.

As used herein, a “biological sample” (or just “sample”) can comprise,for example, tissue, blood, sera, cells, cell lines, or biologicalfluids such as plasma, interstitial fluid, urine, cerebrospinal fluid,and the like. A biological sample is typically, although notnecessarily, obtained from an individual by a medical practitioner.

As used herein, a “individual” can be a mammalian individual ornon-mammalian individual, preferably a mammalian individual. A mammalianindividual can be a human or non-human, preferably a human. The terms“individual”, “individual”, and “patient” are used hereininterchangeably.

A “healthy” or “normal” individual or biological sample is a individualor biological sample in which the disease of interest (e.g., lungcancer) is not detectable, as ascertained by using conventionaldiagnostic methods (such a biological sample can interchangeably bereferred to as a “control” sample).

As used herein, “disease(s)” include cancer, especially aerodigestivecancers, and particularly lung cancer, as well as associated diseasesand pathologies, such as other lung diseases.

The term “diagnose” (or “diagnosing”, etc.) refers to determining thecurrent state or status (e.g., the presence/absence or characteristics)of a disease condition, such as initially detecting the presence of adisease, characterizing/classifying a disease, or detecting diseaseprogression, remission, or recurrence.

The term “prognose” (or “prognosing”, etc.) refers to predicting thefuture course of a disease in a patient who has the disease (e.g.,predicting patient survival).

The term “assess” (or “assessing”, etc.) can encompass “diagnose” and“prognose” but can also encompass making futuredeterminations/predictions about the disease in an individual who doesnot have the disease or determining/predicting the likelihood that adisease will recur in an individual who apparently has been cured of thedisease. The term “assess” can also encompass making assessments of anindividual's response to a therapy, such as predicting whether anindividual is likely to respond favorably to a therapeutic agent or isunlikely to respond to a therapeutic agent (or will experience toxic orother undesirable side effects, for example), selecting a therapeuticagent for administration to an individual, or monitoring or determiningan individual's response to a therapy that has been administered to theindividual.

Thus, “assessing” lung cancer can include, for example, prognosing thefuture course of lung cancer; predicting recurrence of lung cancer in anindividual who apparently has been cured of lung cancer; and/ordetermining or predicting an individual's response to a lung cancertreatment or selecting a lung cancer treatment to administer to anindividual based on the individual's LCM profile (i.e., the differentialabundance level of one or more LCM in the individual).

The following examples may be referred to as either “diagnosing” or“assessing” lung cancer: initially detecting the presence of lungcancer; determining a specific stage, type or sub-type, or otherclassification or characteristic of lung cancer; determining whether alung lesion is a benign lesion or a malignant lung tumor; and/ordetecting/monitoring lung cancer progression (e.g., monitoring lungtumor growth or metastatic spread), remission, or recurrence.

LCM are therefore useful as “prognostic markers” (e.g., predictingdisease progression) and “predictive markers” (e.g., predicting drugresponse), among other uses.

“Treat”, “treating”, or “treatment” of a disease includes: (1)inhibiting the disease, i.e., arresting or reducing the development ofthe disease or its clinical symptoms, or (2) relieving the disease,i.e., causing regression of the disease or its clinical symptom(s).

The term “prophylaxis” is used to distinguish from “treatment,” and toencompass both “preventing” and “suppressing.” It is not always possibleto distinguish between “preventing” and “suppressing,” as the ultimateinductive event or events may be unknown, latent, or the patient is notascertained until well after the occurrence of the event or events.Therefore, the term “protection”, as used herein, is meant to include“prophylaxis.”

A “therapeutically effective amount” means the amount of an agent that,when administered to a individual for treating a disease, is sufficientto effect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on such factors as the agent, the disease andits severity, and the age, weight, etc., of the individual to betreated.

Exemplary embodiments of the invention provide methods for treatingdiseases, especially cancer, and particularly lung cancer, comprisingadministering to a patient a therapeutically effective amount of anantagonist, agonist, or a pharmaceutical composition thereof. Exemplaryembodiments of the invention further provide agonists and antagonists toLCM proteins, as well as pharmaceutical compositions that comprise anagonist or antagonist with a suitable carrier such as a pharmaceuticallyacceptable excipient.

Exemplary agonists or antagonists include antibodies that specificallybind to a LCM protein. Antibodies can be used alone or in combinationwith one or more other therapeutic agents (e.g., as an antibody-drugconjugate or a combination therapy). Further examples of molecules thatcan be used as antagonists include, but are not limited to, smallmolecules that inhibit the function or abundance level of LCM, andinhibitory nucleic acid molecules such as RNAi or antisense nucleic acidmolecules that specifically hybridize to LCM nucleic acid. Exemplaryembodiments of the invention further encompass novel agents identifiedby screening assays using LCM, such as the screening assays describedherein, as well as methods of using these agents, such as for treatmentor diagnostic purposes. For example, an agent identified as describedherein (e.g., a LCM-modulating agent, a LCM-specific nucleic acidmolecule such as an RNAi or antisense molecule, a LCM-specific antibody,a LCM-specific antibody-drug conjugate, or a LCM-binding partner) can beused in an animal or other model, such as to determine efficacy,toxicity, or side effects of treatment with the agent.

Modulators of LCM protein activity, such as modulators identifiedaccording to the drug screening assays described herein, can be used totreat a individual with a disorder mediated by a LCM, e.g., by treatingcells or tissues that express LCM at a differential level. Methods oftreatment can include the step of administering a modulator of LCMactivity in a pharmaceutical composition to a individual in need of suchtreatment.

In certain exemplary embodiments, if decreased expression or activity ofa protein is desired, an antibody to the protein or aninhibitor/antagonist and the like, or a pharmaceutical agent containingone or more of these molecules, can be administered to an individual. Inother exemplary embodiments, if increased expression or activity of aprotein is desired, the protein itself or an agonist/enhancer and thelike, or a pharmaceutical agent containing one or more of thesemolecules, can be administered. Administration can be effected bymethods well known in the art and may include delivery by an antibodyspecifically targeted to the protein. Neutralizing antibodies, whichinhibit dimer formation, can be used when decreased expression oractivity of a protein is desired.

Although modulating agents can be administered in a pure orsubstantially pure form, modulating agents can also be administered aspharmaceutical compositions, formulations, or preparations with acarrier. Exemplary formulations of the invention, such as for human orveterinary use, comprise a suitable active LCM-modulating agent,together with one or more pharmaceutically acceptable carriers and,optionally, other therapeutic ingredients. The carrier(s) are“acceptable” in the sense of being compatible with other ingredients ofa formulation and not deleterious to the recipient thereof. Theformulations can be presented in unit dosage form and can be prepared byany method known to the skilled artisan.

Examples of suitable pharmaceutical carriers include proteins such asalbumins (e.g., U.S. Pat. No. 4,507,234), peptides and polysaccharidessuch as aminodextran (e.g., U.S. Pat. No. 4,699,784), and water. Acarrier can also bear an agent by noncovalent bonding or byencapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos.4,429,008 and 4,873,088). Carriers specific for radionuclide agentsinclude radiohalogenated small molecules and chelating compounds. Forexample, U.S. Pat. No. 4,735,792 discloses representativeradiohalogenated small molecules and their synthesis. A radionuclidechelate can be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, metal oxide, radionuclide. For example, U.S. Pat. No. 4,673,562discloses representative chelating compounds and their synthesis.

Methods of preparing pharmaceutical formulations typically include thestep of bringing into association the active ingredient with thecarrier, which constitutes one or more accessory ingredients.Formulations can be prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers, or both, and then, if necessary, shaping the productinto the desired formulation.

Formulations suitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration can comprise sterile aqueous solutions ofthe active ingredient with solutions, which can be isotonic with theblood of the recipient. Such formulations can be prepared by dissolvingsolid active ingredient in water containing physiologically compatiblesubstances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and thelike, and having a buffered pH compatible with physiological conditionsto produce an aqueous solution, and rendering the solution sterile.These may be present in unit or multi-dose containers, for example,sealed ampoules or vials.

Exemplary formulations of the invention can incorporate a stabilizer.Exemplary stabilizers include polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, detergents, and organicacids, which can be used either alone or as admixtures. Thesestabilizers can be incorporated in an amount of, for example,0.11-10,000 parts by weight per part by weight of an agent. If two ormore stabilizers are to be used, their total amount can be within therange specified above. These stabilizers can be used in aqueoussolutions at an appropriate concentration and pH. The specific osmoticpressure of such aqueous solutions can be in the range of 0.1-3.0osmoles, preferably in the range of 0.8-1.2. The pH of the aqueoussolution can be adjusted to be within the range of 5.0-9.0, preferablywithin the range of 6-8. In formulating an antibody or antibody-drugconjugate, an anti-adsorption agent can be used.

Additional pharmaceutical methods can be employed to control duration ofaction. Controlled release can be achieved through the use of polymer tocomplex or absorb the proteins or their derivatives. Controlled deliverycan be achieved by selecting appropriate macromolecules (e.g.,polyester, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe methods of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled-releasepreparations is to incorporate an anti-LCM antibody into particles of apolymeric material such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively,instead of incorporating these agents into polymeric particles, it ispossible to entrap these materials in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions can be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic, among others.

Any of the therapeutic agents provided herein may be administered incombination with other therapeutic agents. Selection of agents for usein combination therapy can be made by one of ordinary skill in the artaccording to conventional pharmaceutical principles. A combination oftherapeutic agents may act synergistically to affect treatment of aparticular disorder at a lower dosage of each agent.

7. Methods of Detection and Diagnosis Based on LCM Proteins

LCM proteins are useful for diagnosing a disease, particularly diseasesin which the protein is over- or under-expressed, especially cancer, andparticularly lung cancer. The diagnostic methods may be further suitablefor monitoring disease progression in patients undergoing treatment, orfor testing for reoccurrence of disease in patients who were previouslytreated for a disease, for example. Accordingly, exemplary embodimentsof the invention provide methods for detecting the presence of, orabundance levels of, a LCM protein in a biological sample.

In vitro techniques for detection of proteins include, but are notlimited to, enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, a proteincan be detected in vivo in a individual by introducing into theindividual a labeled antibody (or other types of detection agent)specific for the protein marker. For example, an antibody can be labeledwith a radioactive marker whose presence and location in a individualcan be detected by standard imaging techniques. Also useful are methodsthat detect variants of a protein (e.g., allelic variants or mutations)and methods that detect fragments of a protein in a sample.

Examples of immunoassays that can be used in accordance with exemplaryembodiments of the invention include, but are not limited to,competitive and non-competitive assays using techniques such as Westernblots, radioimmunoassays, ELISA, “sandwich” immunoassays,immunoprecipitation assays, precipitation reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays, as well as fluorescencepolarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzymeimmunoassay (EIA), and nephelometric inhibition immunoassay (NIA).Immunoassays such as these are well known in the art and are describedin, for example, Ausubel et al., Current Protocols in Molecular Biology,1992-2006.

For example, ELISA can be used to detect or quantify one or more LCM.For example, ELISA (or other types of LCM assays) can be used to detectLCM in, for example, a high-risk individual or population, in anindividual suspected of having lung cancer, or in an individual with nosuspicion of having lung cancer (e.g., an individual undergoing routinescreening for lung cancer).

In certain exemplary ELISA methods, an antibody that specifically bindsto an LCM antigen may be coated to the well of a suitable container(e.g., a 96 well microtiter plate), a patient sample (e.g., a serumsample) can be added to the well and incubated for a period of time, andthe presence of the LCM antigen in the patient sample can be detectedupon binding of the LCM antigen in the patient sample to the antibodythat is coated to the well. In this instance, a second antibodyconjugated to a detectable moeity may optionally be added following theaddition of the patient sample to the coated well. ELISA methods such asthese may be modified or optimized as desired.

Further, instead of coating the well with an antibody to an LCM antigen,the LCM antigen itself may be coated to the well. Thus, in certainexemplary ELISA methods, an LCM antigen can be coated to the well of asuitable container (e.g., a 96 well microtiter plate), an antibody(which may optionally be conjugated to a detectable moiety such as anenzymatic substrate like horseradish peroxidase or alkaline phosphatase)to the LCM antigen can be added to the well and incubated for a periodof time, and the presence of the LCM antigen can be detected. Theantibody to the LCM antigen does not have to be conjugated to adetectable moiety; for example, a second antibody (which recognizes theantibody to the LCM antigen) conjugated to a detectable moeity may beadded to the well. ELISA methods such as these may be modified oroptimized as desired.

Proteins can be isolated from a biological sample (such as from apatient having a disease) and assayed for the presence of a mutation. Amutation can include, for example, one or more amino acid substitutions,deletions, insertions, rearrangements (such as from aberrant splicingevents), or inappropriate post-translational modifications. Examples ofanalytic methods useful for detecting mutations in a protein include,but are not limited to, altered electrophoretic mobility, alteredtryptic peptide digest, altered protein activity in cell-based orcell-free assays, alteration in substrate or antibody-binding patterns,altered isoelectric point, and direct amino acid sequencing.

Information obtained by detecting a protein can be used, for example, todetermine prognosis and appropriate course of treatment for a disease.For example, individuals with a particular LCM expression level or stageof disease may respond differently to a given treatment that individualslacking LCM expression, or individuals over- or under-expressing LCM.Information obtained from diagnostic methods of the invention canprovide for the personalization of diagnosis and treatment.

In exemplary embodiments, the invention provides methods for diagnosingdisease (including, for example, monitoring treatment response orrecurrence of disease following treatment) in a individual comprising:determining the abundance level of LCM (e.g., LCM protein or nucleicacid, or protein or nucleic acid fragments thereof) in a test samplefrom the individual; wherein a difference in the abundance level of LCMrelative to the abundance level of LCM in a test sample from a healthyindividual, or the level established for a healthy individual, isindicative of disease.

Exemplary embodiments of the invention provide methods for diagnosingdiseases having differential protein expression. For example, normal,control, or standard values (e.g., that represent typical expressionlevels of a protein in healthy individuals) can be established, such asby combining body fluids, tissues, or cell extracts taken from a normalhealthy mammalian or human individual with specific antibodies to aprotein under conditions for complex formation. Standard values forcomplex formation in normal and disease tissues can be established byvarious methods, such as photometric means. Complex formation, as it isexpressed in a test sample, can be compared with the standard values.Deviation from a normal standard and toward a disease standard canprovide parameters for disease diagnosis or prognosis while deviationaway from a disease standard and toward a normal standard can be used toevaluate treatment efficacy, for example.

Immunological methods for detecting and measuring complex formation as ameasure of protein expression using either specific polyclonal ormonoclonal antibodies are known in the art. Examples of such techniquesinclude ELISAs, radioimmunoassays (RIAs), flow cytometry (also referredto as fluorescence-activated cell sorting, or FACS), and antibodyarrays. Such immunoassays typically involve the measurement of complexformation between a protein and its specific antibody. These assays andtheir quantitation against purified, labeled standards are well known inthe art (Ausubel, supra, unit 10.1-10.6). For example, a two-site,monoclonal-based immunoassay utilizing antibodies reactive to twonon-interfering epitopes can be utilized, and competitive binding assaycan also be utilized (Pound (1998) Immunochemical Protocols, HumanaPress, Totowa N.J.).

For diagnostic applications, an antibody can be labeled with adetectable moiety (interchangeably referred to as a “label” or“detectable substance”), such as to facilitate detection by variousimaging methods. Methods for detection of labels include, but are notlimited to, fluorescence, light, confocal, and electron microscopy;magnetic resonance imaging and spectroscopy; fluoroscopy, computedtomography and positron emission tomography. Examples of suitable labelsinclude, but are not limited to, fluorescein, rhodamine, eosin and otherfluorophores, radioisotopes, gold, gadolinium and other lanthanides,paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, labels may be bi- or multi-functional andbe detectable by more than one of the methods listed. Antibodies may bedirectly or indirectly labeled. Attachment of labels to antibodiesincludes covalent attachment of a label, incorporation of a label intoan antibody, and covalent attachment of a chelating compound for bindingof a label, among others well known in the art.

Numerous detectable moieties are available for labeling antibodies,including, but not limited to, those in the following categories:

(a) Radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, and ¹³¹I. An antibody can belabeled with a radioisotope using the techniques described in CurrentProtocols in Immunology, vol 1-2, Coligen et al., Ed.,Wiley-Interscience, New York, Pubs. (1991-2006), for example, andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available.Fluorescent labels can be conjugated to an antibody using the techniquesdisclosed in Current Protocols in Immunology, supra, for example.Fluorescence can be quantified using a fluorometer.

(c) Various enzyme-substrate labels are available (e.g., U.S. Pat. Nos.4,275,149 and 4,318,980). An enzyme generally catalyzes a chemicalalteration of a chromogenic substrate which can be measured usingvarious techniques. For example, an enzyme may catalyze a color changein a substrate, which can be measured spectrophotometrically.Alternatively, an enzyme may alter the fluorescence or chemiluminescenceof a substrate. Techniques for quantifying a change in fluorescence aredescribed herein and well known in the art A chemiluminescent substratebecomes electronically excited by a chemical reaction and may then emitlight which can be measured (using a chemiluminometer, for example) ordonates energy to a fluorescent acceptor. Examples of enzymatic labelsinclude luciferases (e.g., firefly luciferase and bacterial luciferase;U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,malate dehydrogenase, urease, peroxidase such as horseradish peroxidase(HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to antibodies are described inO'Sullivan et al., Methods for the Preparation of Enzyme-AntibodyConjugates for Use in Enzyme Immunoassay, in Methods in Enzyme. (Ed. J.Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

A label can be indirectly conjugated with an antibody. The skilledartisan will be aware of various techniques for achieving this. Forexample, an antibody can be conjugated with biotin and any of the threebroad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of a label with anantibody, an antibody can be conjugated with a small hapten (e.g.,digoxin) and one of the different types of labels mentioned above can beconjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).Thus, indirect conjugation of a label with an antibody can be achieved.

Antibodies can be used to isolate LCM proteins by standard techniques,such as affinity chromatography or immunoprecipitation, and antibodiescan facilitate the purification of the natural protein from cells andrecombinantly-produced protein expressed in host cells. Biologicalsamples can be tested directly for the presence of a LCM protein byassays (e.g., ELISA or radioimmunoassay) and format (e.g., microwells,dipstick, etc., as described in International Patent Publication WO93/03367). Alternatively, proteins in a sample can be size separated(e.g., by polyacrylamide gel electrophoresis (PAGE)), in the presence orabsence of sodium dodecyl sulfate (SDS), and the presence of a LCMdetected by immunoblotting (e.g., Western blotting).

Antibody binding can also be detected by “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitation reactions,immunodiffusion assays, in situ immunoassays (e.g., using colloidalgold, enzyme or radioisotope labels, for example), precipitationreactions, agglutination assays (e.g., gel agglutination assays,hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.

In certain exemplary embodiments, antibody binding can be detected bydetecting a label on the primary antibody. In other exemplaryembodiments, a primary antibody can be detected by detecting binding ofa secondary antibody or reagent to the primary antibody. In furtherexemplary embodiments, the secondary antibody is labeled. Numerous meansare known in the art for detecting binding in an immunoassay and arewithin the scope of the invention. In some embodiments, an automateddetection assay is utilized. Methods for the automation of immunoassaysare well known in the art (e.g., U.S. Pat. Nos. 5,885,530: 4,981,785:6,159,750: and 5,358,691, each of which is herein incorporated byreference). In some embodiments, the analysis and presentation ofresults are also automated. For example, in some embodiments, softwarethat generates a prognosis based on the presence or absence of one ormore antigens can be implemented.

Competitive binding assays typically rely on the ability of a labeledstandard to compete with a test sample for binding with a limited amountof antibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies can be separated from the standard and testsample that remain unbound.

Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected. In typical sandwich assays, the test sampleto be analyzed is bound by a first antibody, which is immobilized on asolid support, and thereafter a second antibody binds to the testsample, thus forming an insoluble three-part complex (e.g., U.S. Pat.No. 4,376,110). The second antibody can itself be labeled with adetectable moiety (direct sandwich assays) or can be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one type of sandwich assay is anELISA assay, in which case the detectable moiety is an enzyme.

Antibodies can also be used for in vivo diagnostic assays. Generally, anantibody can be labeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C,¹³¹I, ³H, ³²P or ³⁵S) so that disease cells or tissues can be localizedusing immunoscintiography, for example. In certain embodiment,antibodies or fragments thereof bind to the extracellular domains of twoor more LCM proteins and the affinity value (Kd) is less than 1×10⁸ M.

For immunohistochemistry, a disease tissue sample may be, for example,fresh or frozen or may be embedded in paraffin and fixed with apreservative such as formalin. A fixed or embedded section can becontacted with a labeled primary antibody and secondary antibody,wherein the antibody is used to detect LCM protein expression in situ.

Antibodies can be used to detect a marker protein in situ, in vitro, orin a cell lysate or supernatant in order to evaluate the abundance andpattern of expression. Also, antibodies can be used to assess abnormaltissue distribution or abnormal expression during development orprogression of a biological condition. Antibodies against LCM proteinsare useful for detecting the presence of the proteins in cells ortissues to determine the pattern of expression of the proteins amongvarious tissues in an organism and over the course of the organism'sdevelopment.

Further, antibodies can be used to assess expression in disease statessuch as in active stages of a disease or in an individual with apredisposition toward disease related to the protein's function. When adisorder is caused by inappropriate tissue distribution, developmentalexpression, or level of expression of a protein, or expressed/processedform, for example, an antibody can be prepared against the normalprotein. If a disorder is characterized by a specific mutation in aprotein, antibodies specific for the mutant protein can be used to assayfor the presence of the specific mutant protein and to target the mutantprotein for therapeutic purposes. Antibodies are also useful asdiagnostic tools, as immunological markers for aberrant protein analyzedby electrophoretic mobility, isoelectric point, tryptic peptide digest,and other physical assays known in the art.

Certain exemplary diagnostic methods of the invention can also includemonitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting, for example, the function, activity,expression level, tissue distribution, or developmental expression of aprotein, antibodies directed against the protein can be used to monitortherapeutic efficacy and to modify a treatment regimen as necessary.

Additionally, antibodies to a marker protein are useful inpharmacogenomic analysis. For example, antibodies prepared againstpolymorphic proteins can be used to identify individuals that requiremodified treatment modalities. Moreover, the marker proteins andantibodies thereto can be used for clinical trials, such as to identifyindividuals that should be included (e.g., individuals more likely torespond to a therapy) or excluded (e.g., individuals less likely torespond to a therapy, or individuals more likely to experience harmfulside effects from a therapy) from a clinical trial.

The invention also encompasses kits for using antibodies to detect thepresence of a marker protein in a biological sample. An exemplary kitcan comprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be configured to detect a single marker proteinor epitope or can be configured to detect one of a multitude ofepitopes, such as in an antibody detection array.

LC/MS and ICAT

In certain exemplary embodiments, the invention provides detection ordiagnostic methods of a LCM by using LC/MS. Proteins can be preparedfrom cells by methods known in the art (e.g., Zhang et al., NatureBiotechnology 21(6):660-666 (2003)). The differential expression ofproteins in disease and healthy (or drug-resistant and drug-sensitive,for example) samples can be quantitated using mass spectrometry and ICAT(Isotope Coded Affinity Tag) labeling, which is known in the art. ICATis an isotope label technique that allows for discrimination between twopopulations of proteins, such as a healthy and a disease sample.Over-expression or under-expression of a LCM protein, as measured byICAT, can indicate, for example, the likelihood of having or developinga disease or an associated pathology.

LC/MS spectra can be collected for labeled samples and processed asfollows. The raw scans from the LC/MS instrument can be individualed topeak detection and noise reduction software. Filtered peak lists canthen be used to detect ‘features’ corresponding to specific peptidesfrom the original sample(s). Features are characterized by theirmass/charge ratio, charge, retention time, isotope pattern, and/orintensity, for example.

The intensity of a peptide present in both healthy and disease samplescan be used to calculate the differential expression, or relativeabundance, of the peptide. The intensity of a peptide found exclusivelyin one sample can be used to calculate a theoretical expression ratiofor that peptide (singleton). Expression ratios can be calculated foreach peptide in an assay or experiment.

Statistical tests can be performed to assess the robustness of the dataand select statistically significant differentials. To ensure theaccuracy of data, the following steps can be taken: a) ensure thatsimilar features are detected in all replicates of an experiment; b)assess the distribution of the log ratios of all peptides (a Gaussian isexpected); c) calculate the overall pair wise correlations between ICATLC/MS maps to ensure that the expression ratios for peptides arereproducible across multiple replicates; and d) aggregate multipleexperiments in order to compare the expression ratio of a peptide inmultiple diseases or disease samples.

8. Methods of Treatment Based on LCM Proteins

a. Antibody Therapy

Antibodies of the invention can be used for therapeutic purposes. It iscontemplated that antibodies of the invention may be used to treat amammal, preferably a human, with a disease, especially cancer, andparticularly lung cancer. The antibodies can be delivered alone, in apharmaceutical composition (such as with a carrier), or conjugated toone or more therapeutic agents, for example.

Antibodies can be useful for modulating (e.g., agonizing orantagonizing) protein function, such as for therapeutic purposes.Antibodies can also be useful for inhibiting protein function by, forexample, blocking the binding of a LCM protein to a binding partner suchas a substrate, which can be useful therapeutically. Antibodies can beprepared against, for example, specific portions of a protein thatcontain domains required for protein function, or against intact proteinthat is associated with a cell membrane.

Antibodies of the invention can also be used for enhancing the immuneresponse. The antibodies can be administered in amounts similar to thoseused for other therapeutic administrations of antibodies. For example,pooled gamma globulin can be administered at a range of about 1 mg toabout 100 mg per patient.

Antibodies reactive with LCM proteins can be administered alone or inconjunction with other therapies, such as anti-cancer therapies, to amammal afflicted with cancer or other disease. Examples of anti-cancertherapies include, but are not limited to, chemotherapy, radiationtherapy, and adoptive immunotherapy therapy with TIL (tumor infiltratinglymphocytes).

The selection of an antibody subclass for therapy may depend upon thenature of the antigen to be acted upon. For example, an IgM may bepreferred in situations where the antigen is highly specific for thedisease marker and rarely occurs on normal cells. However, where thedisease-associated antigen is also expressed in normal tissues, althoughat lower levels, the IgG subclass may be preferred. The IgG subclass maybe preferred in these instances because the binding of at least two IgGmolecules in close proximity is typically required to activatecomplement, and therefore less complement-mediated damage may occur innormal tissues that express smaller amounts of the antigen and thus bindfewer IgG antibody molecules. Furthermore, IgG molecules, by beingsmaller, may be more able than IgM molecules to localize to a diseasedtissue.

A mechanism for antibody therapy can be that a therapeutic antibodyrecognizes a soluble or cell surface marker protein that is expressed(preferably, over-expressed) in a disease cell. By NK cell or complementactivation, or conjugation of the antibody with an immunotoxin orradiolabel, the interaction of the antibody with the marker protein canabrogate ligand/receptor interaction or activation of apoptosis, forexample.

Potential mechanisms of antibody-mediated cytotoxicity of diseased cellsinclude phagocyte (antibody-dependent cellular cytotoxicity (ADCC)),complement (complement-dependent cytotoxicity (CDC)), naked antibody(receptor cross-linking apoptosis and growth factor inhibition), ortargeted payload labeled with a therapeutic agent, such as aradionuclide, immunotoxin, or immunochemotherapeutic or othertherapeutic agent.

In certain exemplary embodiments, an antibody is administered to anonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents, and other mammals in which preclinicalstudies are performed. Such mammals may be established animal models fora disease or may be used to study toxicity of an antibody of interest,for example. Dose escalation studies may be performed in the mammal, forexample.

An antibody can be administered to an individual by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunomodulatory treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, an antibody can beadministered by pulse infusion, particularly with declining doses of theantibody. The dosing can be given by injections, such as intravenous orsubcutaneous injections, which may depend in part on whether theadministration is brief or chronic.

For the prevention or treatment of a disease, the appropriate dosage ofan antibody may depend on the type of disease to be treated, theseverity and the course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician.

Depending on the type and severity of disease, about 1 μg/kg to 150mg/kg (e.g., 0.1-20 mg/kg) of antibody can be an initial candidatedosage for administration to a patient, whether, for example, by one ormore separate administrations, or by continuous infusion. A typicaldaily dosage may range from about 1 μg/kg to 100 mg/kg or more,depending on such factors as those mentioned above. An antibody-drugconjugate can be administered from about 1 μg/kg to 50 mg/kg, typicallyfrom about 0.1-20 mg/kg, whether, for example, by one or more separateadministrations, or by continuous infusion. A typical daily dosage mayrange from about 0.1 mg/kg to 10 mg/kg, or from about 0.3 mg/kg to about7.5 mg/kg, depending on such factors as those mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment can be sustained until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be useful.Therapy progress can be monitored by conventional techniques and assays.

Antibody composition can be formulated, dosed, and administered in amanner consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

An antibody may optionally be formulated with, or administered with, oneor more therapeutic agents used to prevent or treat the disorder inquestion. For example, an antibody can be administered as a co-therapywith a standard of care therapeutic for the specific disease beingtreated.

b. Other Immunotherapy

An “immunogenic peptide” is a peptide that comprises an allele-specificmotif such that the peptide typically will bind an MHC allele (HLA inhuman) and be capable of inducing a CTL (cytotoxic T-lymphocytes)response. Thus, immunogenic peptides typically are capable of binding toan appropriate class I or II MHC molecule and inducing a cytotoxic Tcell or T helper cell response against the antigen from which theimmunogenic peptide is derived.

Peptides derived from a LCM protein can be modified to increase theirimmunogenicity, such as by enhancing the binding of the peptide to theMHC molecules in which the peptide is presented. The peptide or modifiedpeptide can be conjugated to a carrier molecule to enhance theantigenicity of the peptide. Examples of carrier molecules, include, butare not limited to, human albumin, bovine albumin, lipoprotein andkeyhole limpet hemo-cyanin (“Basic and Clinical Immunology” (1991)Stites and Terr (eds) Appleton and Lange, Norwalk Conn., San Mateo,Calif.).

Further, amino acid sequence variants of a peptide can be prepared, suchas by altering the nucleic acid sequence of the DNA which encodes thepeptide, or by peptide synthesis. At the genetic level, these variantscan be prepared by, for example, site-directed mutagenesis ofnucleotides in the DNA encoding the peptide, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. The variants can exhibit the same qualitative biologicalactivity as the nonvariant peptide.

Exemplary embodiments of the invention provide peptides or modifiedpeptides derived from a LCM protein that are differentially expressed indisease. Examples of peptide modifications include, but are not limitedto, substitutions, deletions, or additions of one or more amino acids ina given immunogenic peptide sequence, or mutation of existing aminoacids within a given immunogenic peptide sequence, or derivatization ofexisting amino acids within a given immunogenic peptide sequence. Anyamino acid in an immunogenic peptide sequence may be modified. In someembodiments, at least one amino acid can be substituted or replacedwithin the given immunogenic peptide sequence. Any amino acid may beused to substitute or replace a given amino acid within the immunogenicpeptide sequence. Modified peptides can include any immunogenic peptideobtained from differentially expressed proteins, which has been modifiedand exhibits enhanced binding to the MHC molecule with which itassociates when presented to a T-cell. These modified peptides can besynthetically or recombinantly produced by conventional methods, forexample.

In certain exemplary embodiments of the invention, the peptidescomprise, or consist of, sequences of about 5-30 amino acids in lengthwhich are immunogenic (i.e., capable of inducing an immune response wheninjected into a individual).

In certain exemplary embodiments, the peptides may be used, for example,to treat T cell-mediated pathologies. The term “T cell-mediatedpathologies” refers to any condition in which an inappropriate T cellresponse is a component of the pathology. The term is intended toencompass both T cell mediated diseases and diseases resulting fromunregulated clonal T cell replication.

Modified (e.g., recombinant) or natural LCM proteins, or fragmentsthereof, can be used as a vaccine either prophylactically ortherapeutically. When provided prophylactically, a vaccine can beprovided in advance of any evidence of disease. The prophylacticadministration of a disease vaccine may serve to prevent or attenuate adisease in a mammal such as a human.

An exemplary vaccine formulation can comprise an immunogen that inducesan immune response directed against a disease-associated antigen such asa LCM protein. For example, a substantially or partially purified LCMprotein or fragments thereof can be administered as a vaccine in apharmaceutically acceptable carrier. An immunogen can be administered ina pure or substantially pure form, or can be administered as apharmaceutical composition, formulation, or preparation. Exemplary dosesof protein that can be administered are about 0.001 to about 100 mg perpatient, or about 0.01 to about 100 mg per patient. Immunization can berepeated as necessary until a sufficient titer of anti-immunogenantibody or immune cells has been obtained.

Vaccine can be prepared using, for example, recombinant protein orexpression vectors comprising a nucleic acid sequence encoding all orpart of a LCM protein. Examples of vectors that can be used in vaccinesinclude, but are not limited to, defective retroviral vectors,adenoviral vectors vaccinia viral vectors, fowl pox viral vectors, orother viral vectors (Mulligan, R. C., (1993) Science 260:926-932). Thevectors can be introduced into a mammal (e.g., a human) either prior toany evidence of a disease or to mediate regression of a disease in amammal afflicted with the disease. Examples of methods for administeringa viral vector into mammals include, but are not limited to, exposure ofcells to the virus ex vivo, or injection of the retrovirus or a producercell line of the virus into the affected tissue, or intravenousadministration of the virus. Alternatively, the vector can beadministered locally by direct injection into a disease lesion ortopical application in a pharmaceutically acceptable carrier. Thequantity of viral vector to be administered can be based on the titer ofvirus particles. An exemplary range can be about 10⁶ to about 10¹¹ virusparticles per mammal.

After immunization, the efficacy of the vaccine can be assessed by, forexample, the production of antibodies or immune cells that recognize theantigen, as assessed by specific lytic activity, specific cytokineproduction, or disease regression, which can be measured usingconventional methods. If the mammal to be immunized is already afflictedwith a disease, the vaccine can be administered in conjunction withother therapeutic treatments. Examples of other therapeutic treatmentsinclude, but are not limited to, adoptive T cell immunotherapy andcoadministration of cytokines or other therapeutic drugs.

In certain embodiments, mammals, preferably humans, at high risk fordisease, especially cancer, are prophylactically treated with vaccinesof the invention. Examples include, but are not limited to, individualswith a family history of a disease, individuals who themselves have ahistory of disease (e.g., cancer that has been previously resected andat risk for reoccurrence), or individuals already afflicted with adisease. When provided therapeutically, a vaccine can be provided toenhance the patient's own immune response to a disease antigen. Anexemplary vaccine, which acts as an immunogen, can be a cell, celllysate from cells transfected with a recombinant expression vector, or aculture supernatant containing the expressed protein, for example.Alternatively, an immunogen can be, for example, a partially orsubstantially purified recombinant protein, peptide, or analog thereof,or a modified protein, peptide, or analog thereof. The proteins orpeptides can be, for example, conjugated with lipoprotein oradministered in liposomal form or with adjuvant.

Vaccination can be carried out using conventional methods. For example,an immunogen can be used in a suitable diluent such as saline or water,or complete or incomplete adjuvants. Further, an immunogen may or maynot be bound to a carrier, including carriers to increase theimmunogenicity of the immunogen. Examples of carrier molecules include,but are not limited to, bovine serum albumin (BSA), keyhole limpethemocyanin (KLH), tetanus toxoid, and the like. An immunogen also may becoupled with lipoproteins or administered in liposomal form or withadjuvants. An immunogen can be administered by any route appropriate forantibody production such as intravenous, intraperitoneal, intramuscular,subcutaneous, and the like. An immunogen can be administered once or atperiodic intervals until a significant titer of anti-LCM immune cells oranti-LCM antibody is produced. The presence of anti-LCM immune cells canbe assessed by measuring the frequency of precursor CTL (cytotoxicT-lymphocytes) against LCM antigen prior to and after immunization by aCTL precursor analysis assay (Coulie et al., 1992, International JournalOf Cancer 50:289-297). An immunoassay can be used to detect antibody inserum.

The safety of a vaccine can be determined by examining the effect ofimmunization on the general health of an immunized animal (e.g., weightchange, fever, change in appetite or behavior, etc.) and looking forpathological changes during autopsies. After initial testing in animals,a vaccine can be tested in patients having a disease of interest.Conventional methods can be used to evaluate the immune response of apatient to determine the efficiency of the vaccine.

In certain exemplary embodiments of the invention, a LCM protein orfragments thereof, or a modified LCM protein, can be exposed todendritic cells cultured in vitro. The cultured dendritic cells providea means of producing T-cell dependent antigens comprised of dendriticcell-modified antigen or dendritic cells pulsed with antigen, in whichthe antigen is processed and expressed on the antigen-activateddendritic cell. The antigen-activated dendritic cells or processeddendritic cell antigens can be used as immunogens for vaccines or forthe treatment of diseases. The dendritic cells can be exposed to theantigen for sufficient time to allow the antigens to be internalized andpresented on the surface of dendritic cells. The resulting dendriticcells or the dendritic cell-processed antigens can then be administeredto an individual in need of therapy. Such methods are described inSteinman et al. (WO93/208185) and in Banchereau et al. (EPO Application0563485A1).

In certain exemplary embodiments of the invention, T-cells isolated fromindividuals can be exposed to a LCM protein or fragment thereof, or amodified LCM protein, in vitro and then administered in atherapeutically effective amount to a patient in need of such treatment.Examples of where T-lymphocytes can be isolated include, but are notlimited to, peripheral blood cells lymphocytes (PBL), lymph nodes, ortumor infiltrating lymphocytes (TIL). Such lymphocytes can be isolatedfrom the individual to be treated or from a donor by methods known inthe art and cultured in vitro (Kawakami et al., 1989, J. Immunol. 142:2453-3461). Lymphocytes can be cultured in media such as RPMI or RPMI1640 or AIM V for 1-10 weeks. Viability can be assessed by trypan bluedye exclusion assay. Examples of how these sensitized T-cells can beadministered to a mammal include, but are not limited to, intravenously,intraperitoneally, or intralesionally. Parameters that can be assessedto determine the efficacy of these sensitized T-lymphocytes include, butare not limited to, production of immune cells in the mammal beingtreated or tumor regression. Conventional methods can be used to assessthese parameters. Such treatment can be given in conjunction withcytokines or gene-modified cells, for example (Rosenberg et al., 1992,Human Gene Therapy, 3: 75-90; Rosenberg et al., 1992, Human GeneTherapy, 3: 57-73).

9. Screening Methods Using LCM Proteins

Exemplary embodiments of the invention provide methods of screening foragents (interchangeably referred to by such terms as candidate agents,compounds, or candidate compounds) that modulate LCM protein activity(interchangeably referred to as protein function). Examples of candidateagents include, but are not limited to, proteins, peptides, antibodies,nucleic acids (such as antisense and RNAi nucleic acid molecules), andsmall molecules. Exemplary embodiments of the invention further provideagents identified by these screening methods, and methods of using theseagents, such as for treating diseases, especially cancer, andparticularly lung cancer.

Exemplary screening methods can typically comprise the steps of (i)contacting a LCM protein with a candidate agent, and (ii) assaying forLCM protein activity, wherein a change in protein activity in thepresence of the agent relative to protein activity in the absence of theagent indicates that the agent modulates LCM protein activity.

Other exemplary screening methods can determine a candidate agent'sability to modulate LCM expression. Exemplary methods can typicallycomprise the steps of (i) contacting a candidate agent with a systemthat is capable of expressing LCM protein or LCM mRNA, and (ii) assayingfor the level of LCM protein or LCM mRNA, wherein a change in the levelin the presence of the agent relative to the level in the absence of theagent indicates that the agent modulates LCM expression levels.

Exemplary embodiments of the invention further provide methods to screenfor agents that bind to LCM proteins. Exemplary methods can typicallycomprise the steps of contacting a LCM protein with a test agent andmeasuring the extent of binding of the agent to the LCM protein.

LCM proteins can be used to identify agents that modulate activity of aprotein in its natural state or an altered form that causes a specificdisease or pathology. LCM proteins and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for their ability to bind to LCM. These compounds can befurther screened against functional LCM proteins to determine the effectof the compound on the protein's activity. Further, these compounds canbe tested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) LCM proteins to a desired degree.

LCM proteins can be used to screen agents for their ability to stimulateor inhibit interaction between a LCM protein and a target molecule thatnormally interacts with the LCM protein (e.g., a substrate, anextracellular binding ligand, or a component of a signal pathway that aLCM protein normally interacts with such as a cytosolic signal protein).Exemplary assays can include the steps of combining a LCM protein orfragment thereof with a candidate compound under conditions that allowthe LCM protein (or fragment thereof) to interact with a targetmolecule, and detecting the formation of a complex between the LCMprotein and the target molecule or detecting the biochemical consequenceof the interaction between the LCM protein and the target molecule, suchas any of the associated effects of signal transduction (e.g., proteinphosphorylation, cAMP turnover, adenylate cyclase activation, etc.). Anyof the biological or biochemical functions mediated by a LCM protein canbe used as an endpoint assay to identify an agent that modulates LCMactivity.

Candidate compounds or agents include, but are not limited to, 1)peptides such as soluble peptides, including Ig-tailed fusion peptidesand members of random peptide libraries (see, e.g., Lam et al., Nature354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules (e.g., molecules obtained fromcombinatorial and natural product libraries).

An exemplary candidate compound or agent is a soluble fragment of a LCMthat competes for substrate binding. Other exemplary candidate compoundsinclude mutant LCM proteins or appropriate fragments containingmutations that affect LCM function and thus compete for substrate.Accordingly, a fragment that competes for substrate, for example with ahigher affinity, or a fragment that binds substrate but does not allowrelease, is encompassed by the invention.

Compounds can also be screened by using chimeric proteins in which anyportion of a protein such as an amino terminal extracellular domain, atransmembrane domain (e.g., transmembrane segments or intracellular orextracellular loops), or a carboxy terminal intracellular domain can bereplaced in whole or part by heterologous domains or subregions. Forexample, a substrate-binding region can be used that interacts with adifferent substrate than the substrate that is recognized by a nativemarker protein. Accordingly, a different set of signal transductioncomponents can be available as an end-point assay for activation,thereby allowing assays to be performed in other than the specific hostcell from which a marker is derived.

Competition binding assays can also be used to screen for compounds thatinteract with a marker protein (e.g., binding partners and/or ligands).For example, a test compound can be exposed to a marker protein underconditions that allow the test compound to bind or otherwise interactwith the marker protein. Soluble marker protein can also be added to themixture. If the test compound interacts with the soluble marker protein,it can decrease the amount of complex formed or activity of the markerprotein. This type of assay is particularly useful in instances in whichcompounds are sought that interact with specific regions of a markerprotein. Thus, the soluble marker protein that competes with the markerprotein can contain peptide sequences corresponding to the marker regionof interest.

To perform cell-free drug screening assays, it may be desirable toimmobilize either a LCM protein (or fragment thereof) or a molecule thatbinds the LCM protein (referred to herein as a “binding partner”) tofacilitate separation of complexes from uncomplexed forms, as well as tofacilitate automation of the assays.

Techniques for immobilizing proteins on matrices can be utilized inexemplary drug screening assays. In exemplary embodiments, a fusionprotein can be provided which adds a domain that allows a protein to bebound to a matrix. For example, glutathione-S-transferase fusionproteins can be adsorbed onto glutathione SEPHAROSE beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with cell lysates (e.g., ³⁵S-labeled) and acandidate compound, and the mixture incubated under conditions conduciveto complex formation (e.g., at physiological conditions for salt andpH). Following incubation, the beads can be washed to remove any unboundlabel, and the matrix immobilized and radiolabel determined directly, orin the supernatant after the complexes are dissociated. Alternatively,the complexes can be dissociated from the matrix, separated by SDS-PAGE,and the level of a binding partner found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either a marker protein or a binding partner can be immobilizedby conjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies that are reactive with a markerprotein but do not interfere with binding of the marker protein to itsbinding partner can be derivatized to the wells of a plate, and themarker protein trapped in the wells by antibody conjugation.Preparations of a binding partner and a candidate compound can beincubated in marker protein-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described for GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with abinding partner, or which are reactive with a marker protein and competewith the binding partner, as well as marker protein-linked assays whichrely on detecting an enzymatic activity associated with a bindingpartner.

In exemplary embodiments of the invention, a LCM protein can be used asa “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with a LCM protein and are involved in the protein's activity.The two-hybrid system is based on the modular nature of mosttranscription factors, which typically consist of separable DNA-bindingand activation domains. In exemplary embodiments, the two-hybrid assaycan utilize two different DNA constructs. In one construct, a gene thatencodes a LCM protein can be fused to a gene encoding the DNA bindingdomain of a known transcription factor (e.g., GAL-4). In the otherconstruct, a DNA sequence from a library of DNA sequences that encode anunidentified protein (“prey” or “sample”) can be fused to a gene thatencodes the activation domain of the known transcription factor. If the“bait” and the “prey” proteins are able to interact in vivo, forming aLCM-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ), which can beoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene that encodes the proteinthat interacts with the LCM protein.

Agents that modulate a LCM protein can be identified using one or moreof the above assays, alone or in combination. For example, a cell-basedor cell free system can be used for initial identification of agents,and then activity of the agents can be confirmed in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

10. Diagnosis, Treatment, and Screening Methods Using LCM Nucleic AcidMolecules

The nucleic acid molecules of the invention are useful, for example, asprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as hybridization probes for messengerRNA, transcript/cDNA, and genomic DNA to detect or isolate full-lengthcDNA and genomic clones encoding a LCM protein, or variants thereof. Thenucleic acid molecules are also useful as primers for PCR to amplify anygiven region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence. The nucleic acidmolecules are also useful for producing ribozymes corresponding to all,or a part, of the mRNA produced from the nucleic acid moleculesdescribed herein.

The nucleic acid molecules are also useful for constructing recombinantvectors. Exemplary vectors include expression vectors that express aportion of, or all of, a LCM protein. The nucleic acid molecules arealso useful for expressing antigenic portions of the proteins. Thenucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the proteins. The nucleic acid moleculesare also useful for constructing transgenic animals expressing all, or apart, of the proteins.

A primer or probe can correspond to any sequence along the entire lengthof a LCM-encoding nucleic acid molecule. Accordingly, a primer or probecan be derived from 5′ noncoding regions, coding regions, or 3′noncoding regions, for example.

Exemplary in vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. Exemplary in vitro techniquesfor detecting DNA include Southern hybridizations and in situhybridization. Reverse transcriptase PCR amplification (RT-PCR) and thelike can also be used for detecting RNA expression. A specific exemplarymethod of detection comprises using TaqMan technology (AppliedBiosystems, Foster City, Calif.).

a. Methods of Diagnosis Using Nucleic Acids

Nucleic acid molecules of the invention are useful, for example, ashybridization probes for determining the presence, level, form, and/ordistribution of nucleic acid expression. Exemplary probes can be used todetect the presence of, or to determine levels of, a specific nucleicacid molecule in cells, tissues, and in organisms. Accordingly, probescorresponding to a LCM described herein can be used to assess expressionand/or gene copy number in a given cell, tissue, or organism, which canbe applied to, for example, diagnosis of disorders involving an increaseor decrease in LCM protein expression relative to normal LCM proteinexpression levels.

Probes can be used as part of a diagnostic test kit for identifyingcells or tissues that express LCM protein differentially, such as bymeasuring a level of a LCM-encoding nucleic acid (e.g., mRNA or genomicDNA) in a sample of cells from a individual, or determining if aLCM-encoding nucleic acid is mutated.

Exemplary embodiments of the invention encompass kits for detecting thepresence of LCM-encoding nucleic acid (e.g., mRNA or genomic DNA) in abiological sample. For example, an exemplary kit can comprise reagentssuch as a labeled or labelable nucleic acid or agent capable ofdetecting LCM nucleic acid in a biological sample; means for determiningthe amount of LCM nucleic acid in the sample; and means for comparingthe amount of LCM nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect LCM nucleicacid.

The nucleic acid molecules are useful in diagnostic assays forqualitative changes in LCM nucleic acid expression, and particularly inqualitative changes that lead to pathology. The nucleic acid moleculescan be used to detect mutations in LCM genes and gene expressionproducts such as mRNA. The nucleic acid molecules can be used ashybridization probes to detect naturally occurring genetic mutations ina LCM gene and to determine whether a individual with the mutation is atrisk for a disorder caused by the mutation. Examples of mutationsinclude deletions, additions, or substitutions of one or morenucleotides in a gene, chromosomal rearrangements (such as inversions ortranspositions), and modification of genomic DNA such as aberrantmethylation patterns or changes in gene copy number (such asamplification). Detection of a mutated form of a LCM gene associatedwith a dysfunction can provide a diagnostic tool for an active diseaseor susceptibility to disease in instances in which the disease resultsfrom overexpression, underexpression, or altered expression of a LCMprotein, for example.

Mutations in a LCM gene can be detected at the nucleic acid level by avariety of techniques. For example, genomic DNA, RNA, or cDNA can beanalyzed directly or can be amplified (e.g., using PCR) prior toanalysis. In certain exemplary embodiments, detection of a mutationinvolves the use of a probe/primer in a PCR reaction (see, e.g. U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988) and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in a gene (see Abravaya et al., Nucleic AcidsRes. 23:675-682 (1995)). Exemplary methods such as these can include thesteps of collecting a sample of cells from a patient, isolating nucleicacid (e.g., genomic, mRNA, or both) from the cells of the sample,contacting the nucleic acid with one or more primers which specificallyhybridize to a marker nucleic acid under conditions such thathybridization and amplification of the marker nucleic acid (if present)occurs, and detecting the presence or absence of an amplificationproduct, or detecting the size of the amplification product andcomparing the length to a control sample. Deletions and insertions canbe detected by a change in size of the amplified product compared to anormal genotype. Point mutations can be identified by hybridizingamplified DNA to normal RNA or antisense DNA sequences, for example.

Alternatively, mutations in a LCM gene can be identified, for example,by alterations in restriction enzyme digestion patterns as determined bygel electrophoresis. Further, sequence-specific ribozymes (U.S. Pat. No.5,498,531) can be used to identify the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can be assessed by nucleaseprotection assays such as RNase and 51 protection, or chemical cleavagemethods. Furthermore, sequence differences between a mutant LCM gene anda corresponding wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing diagnostic assays (Naeve, C. W., (1995) Biotechniques19:448), including sequencing by mass spectrometry (e.g., PCTInternational Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in a nucleic acid include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(DGGE) (Myers et al., Nature 313:495 (1985)). Examples of othertechniques for detecting point mutations include selectiveoligonucleotide hybridization, selective amplification, and selectiveprimer extension.

b. Methods of Monitoring Treatment and Pharmacogenomic Methods UsingNucleic Acids

Nucleic acid molecules of the invention are also useful for monitoringthe effectiveness of modulating agents on the expression or activity ofa LCM gene, such as in clinical trials or in a treatment regimen. Forexample, the gene expression pattern of a LCM gene can serve as abarometer for the continuing effectiveness of treatment with a compound,particularly with compounds to which a patient can develop resistance.The gene expression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound. Forexample, based on monitoring nucleic acid expression, the administrationof a compound can be increased or alternative compounds to which thepatient has not become resistant can be administered instead. Similarly,if the level of nucleic acid expression falls below a desirable level,administration of the compound can be commensurately decreased.

The nucleic acid molecules are also useful for testing an individual fora genotype that, while not necessarily causing a disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules provided hereincan be used to assess the mutation content of a marker gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment. For example, marker nucleic acid molecules having geneticvariations that affect treatment can provide diagnostic markers that canbe used to tailor treatment to an individual. Accordingly, theproduction of recombinant cells and animals having these geneticvariations allows effective clinical design of treatment compounds anddosage regimens, for example.

c. Methods of Treatment Using Nucleic Acids

Nucleic acid molecules of the invention are useful to design antisenseconstructs to control LCM gene expression in cells, tissues, andorganisms. An antisense nucleic acid molecule typically blockstranslation of mRNA into LCM protein by hybridizing to marker mRNA in asequence-specific manner. Nucleic acid molecules of the invention canalso be used to specifically suppress gene expression by methods such asRNA interference (RNAi). RNAi and antisense-based gene suppression arewell known in the art (e.g., Science 288:1370-1372, 2000). RNAitypically operates on a post-transcriptional level and is sequencespecific. RNAi and antisense nucleic acid molecules are useful fortreating diseases, especially cancer. RNAi fragments, particularlydouble-stranded (ds) RNAi, as well as antisense nucleic acid moleculescan also be used to generate loss-of-function phenotypes by suppressinggene expression. Accordingly, exemplary embodiments of the inventionprovide RNAi and antisense nucleic acid molecules, and methods of usingthese RNAi and antisense nucleic acid molecules, such as for therapy orfor modulating cell function. Nucleic acid molecules may also beproduced that are complementary to a region of a gene involved intranscription, such as to hybridize to the gene to preventtranscription.

Exemplary embodiments of the invention relate to isolated RNA molecules(double-stranded; single-stranded) that are about 17 to about 29nucleotides (nt) in length, and more particularly about 21 to about 25nt in length, which mediate RNAi (e.g., degradation of mRNA, and suchmRNA may be referred to herein as mRNA to be degraded). With respect toRNAi, the terms RNA, RNA molecule(s), RNA segment(s), and RNAfragment(s) are used interchangeably to refer to RNA that mediates RNAi.These terms include double-stranded RNA, single-stranded RNA, isolatedRNA (e.g., partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA), as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides. Such alterations can include, forexample, addition of non-nucleotide material, such as to the end(s) of a21-25 nt RNA or internally (at one or more nucleotides of the RNA).Nucleotides in exemplary RNA molecules of the invention can alsocomprise non-standard nucleotides, including non-naturally occurringnucleotides or deoxyribonucleotides. Collectively, all such altered RNAsare referred to as analogs or analogs of naturally-occurring RNA. RNA of21-25 nt typically need only be sufficiently similar to natural RNA thatit has the ability to mediate RNAi. As used herein, the phrase “mediatesRNAi” refers to the ability to distinguish which RNAs are to be degradedby RNAi processes. RNA that mediates RNAi directs degradation ofparticular mRNAs by RNAi processes. Such RNA may include RNAs of variousstructures, including short hairpin RNA.

In certain exemplary embodiments, the invention relates to RNA moleculesof about 21 to about 25 nt that direct cleavage of specific mRNA towhich their sequence corresponds. It is not necessary that there be aperfect correspondence (i.e., match) of the sequences, but thecorrespondence must be sufficient to enable the RNA to direct RNAicleavage of the marker mRNA (Holen et al., Nucleic Acids Res.33:4704-4710 (2005)). In an exemplary embodiment, the 21-25 nt RNAmolecules of the invention comprise a 3′ hydroxyl group.

Certain exemplary embodiments of the invention relate to 21-25 nt RNAsof specific genes, produced by chemical synthesis or recombinant DNAtechniques, that mediate RNAi. As used herein, the term “isolated RNA”includes RNA obtained by any means, including processing or cleavage ofdsRNA, production by chemical synthetic methods, and production byrecombinant DNA techniques, for example. Exemplary embodiments of theinvention further relate to uses of the 21-25 nt RNAs, such as fortherapeutic or prophylactic treatment and compositions comprising 21-25nt RNAs that mediate RNAi, such as pharmaceutical compositionscomprising 21-25 nt RNAs and an appropriate carrier.

Further exemplary embodiments of the invention relate to methods ofmediating RNAi of genes of a patient. For example, RNA of about 21 toabout 25 nt which targets a specific mRNA to be degraded can beintroduced into a patient's cells. The cells can be maintained underconditions allowing degradation of the mRNA, resulting in RNA-mediatedinterference of the mRNA of the gene in the cells of the patient.Treatment of cancer patients, for example, with RNAi may inhibit thegrowth and spread of the cancer and reduce tumor size. Treatment ofpatients using RNAi can also be in combination with other therapies. Forexample, RNAi can be used in combination with other treatmentmodalities, such as chemotherapy, radiation therapy, and othertreatments. In an exemplary embodiment, a chemotherapy agent is used incombination with RNAi. In a further exemplary embodiment, GEMZAR(gemcitabine HCl) chemotherapy is used with RNAi.

Treatment of certain diseases by RNAi may require introduction of theRNA into the disease cells. RNA can be directly introduced into a cell,or introduced extracellularly into a cavity, interstitial space, intothe circulation of a patient, or introduced orally, for example.Physical methods of introducing nucleic acids, such as injectiondirectly into a cell or extracellular injection into a patient, may alsobe used. RNA may be introduced into vascular or extravascularcirculation, the blood or lymph system, or the cerebrospinal fluid, forexample. RNA may be introduced into an embryonic stem cell or anothermultipotent cell, which may be derived from a patient. Physical methodsof introducing nucleic acids include injection of a solution containingthe RNA, bombardment by particles covered by the RNA, soaking cells ortissue in a solution of the RNA, or electroporation of cell membranes inthe presence of the RNA. A viral construct packaged into a viralparticle may be used to introduce an expression construct into a cell,with the construct expressing the RNA. Other methods known in the artfor introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, and thelike. The RNA may be introduced along with components that perform oneor more of the following activities: enhance RNA uptake by the cell,promote annealing of the duplex strands, stabilize the annealed strands,or otherwise increase inhibition of the marker gene.

Exemplary RNA of the invention can be used alone or as a component of akit having at least one reagent for carrying out in vitro or in vivointroduction of the RNA to a cell, tissue/fluid, or patient. Exemplarycomponents of a kit include dsRNA and a vehicle that promotesintroduction of the dsRNA. A kit may also include instructions for usingthe kit.

Certain exemplary embodiments of the invention provide compositions andmethods for cleavage of mRNA by ribozymes having nucleotide sequencescomplementary to one or more regions in the mRNA, thereby attenuatingthe translation of the mRNA. Examples of regions in mRNA that can betargeted by ribozymes include coding regions, particularly codingregions corresponding to catalytic or other functional activities of amarker protein, such as substrate binding. These compositions andmethods may be used to treat a disorder characterized by abnormal orundesired marker nucleic acid expression.

In certain exemplary embodiments, nucleic acid molecules of theinvention may be used for gene therapy in individuals having cells thatare aberrant in gene expression of a marker. For example, recombinantcells that have been engineered ex vivo (which can include anindividual's own cells) can be introduced into an individual where thecells produce the desired marker protein to thereby treat theindividual.

d. Methods of Screening Using Nucleic Acids

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate LCM nucleic acid expression.

Exemplary embodiments of the invention thus provide methods foridentifying a compound that can be used to treat a disease associatedwith differential expression of a LCM gene, especially cancer. Exemplarymethods can typically include assaying the ability of a compound tomodulate the expression of a marker nucleic acid to thereby identify acompound that can be used to treat a disorder characterized by undesiredmarker nucleic acid expression. The assays can be performed incell-based or cell-free systems. Examples of cell-based assays includecells naturally expressing marker nucleic acid or recombinant cellsgenetically engineered to express specific marker nucleic acidsequences.

Assays for marker nucleic acid expression can involve direct assay ofmarker nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in a signal pathway. Further, the expression of genesthat are up- or down-regulated in response to a signal pathway can alsobe assayed. In these embodiments, the regulatory regions of these genescan be operably linked to a reporter gene such as luciferase.

Thus, in exemplary embodiments, modulators of gene expression of amarker can be identified in methods wherein a cell is contacted with acandidate agent and the expression of marker mRNA determined. The levelof expression of marker mRNA in the presence of the candidate agent iscompared to the level of expression of marker mRNA in the absence of thecandidate agent. The candidate agent can then be identified as amodulator of marker nucleic acid expression based on this comparison andmay be used, for example, to treat a disorder characterized by aberrantmarker nucleic acid expression. When expression of marker mRNA isstatistically significantly greater in the presence of the candidateagent than in its absence, the candidate agent is identified as astimulator (agonist) of nucleic acid expression. When nucleic acidexpression is statistically significantly less in the presence of thecandidate agent than in its absence, the candidate compound isidentified as an inhibitor (antagonist) of nucleic acid expression.

11. Arrays and Expression Analysis

“Array” (interchangeably referred to as “microarray”) typically refersto an arrangement of at least one, but more typically at least two,nucleic acid molecules, proteins, or antibodies on a substrate. Incertain exemplary arrangements, at least one of the nucleic acidmolecules, proteins, or antibodies typically represents a control orstandard, and other nucleic acid molecules, proteins, or antibodies areof diagnostic or therapeutic interest. In exemplary embodiments, thearrangement of nucleic acid molecules, proteins, or antibodies on thesubstrate is such that the size and signal intensity of each labeledcomplex (e.g., formed between each nucleic acid molecule and acomplementary nucleic acid, or between each protein and a ligand orantibody, or between each antibody and a protein to which the antibodyspecifically binds) is individually distinguishable.

An “expression profile” is a representation of marker expression in asample. A nucleic acid expression profile can be produced using, forexample, arrays, sequencing, hybridization, or amplificationtechnologies for nucleic acids from a sample. A protein expressionprofile can be produced using, for example, arrays, gel electrophoresis,mass spectrometry, or antibodies (and, optionally, labeling moieties)which specifically bind proteins. Nucleic acids, proteins, or antibodiescan be attached to a substrate or provided in solution, and theirdetection can be based on methods well known in the art.

A substrate includes, but is not limited to, glass, paper, nylon orother type of membrane, filter, chip, metal, or any other suitable solidor semi-solid (e.g., gel) support.

Exemplary arrays can be prepared and used according to the methodsdescribed in U.S. Pat. No. 5,837,832; PCT application WO95/11995;Lockhart et al., 1996, Nat. Biotech. 14: 1675-1680; Schena et al., 1996;Proc. Natl. Acad. Sci. 93: 10614-10619; and U.S. Pat. No. 5,807,522.Exemplary embodiments of the invention also provide antibody arrays(see, e.g., de Wildt et al. (2000) Nat. Biotechnol. 18:989-94).

Certain exemplary embodiments of the invention provide a nucleic acidarray for assaying marker expression, which can be composed ofsingle-stranded nucleic acid molecules, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides can be, for example, about 6-60nucleotides in length, about 15-30 nucleotides in length, or about 20-25nucleotides in length.

To produce oligonucleotides to a marker nucleic acid molecule for anarray, the marker nucleic acid molecule of interest is typicallyexamined using a computer algorithm to identify oligonucleotides ofdefined length that are unique to the nucleic acid molecule, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certaininstances, it may be desirable to use pairs of oligonucleotides on anarray. In exemplary embodiments, the “pairs” can be identical, exceptfor one nucleotide (which can be located in the center of the sequence,for example). The second oligonucleotide in the pair (mismatched by one)serves as a control. Any number of oligonucleotide pairs may beutilized.

Oligonucleotides can be synthesized on the surface of a substrate, suchas by using a light-directed chemical process or by using a chemicalcoupling procedure and an ink jet application apparatus (e.g., PCTapplication WO95/251116).

In some exemplary embodiments, an array can be used to diagnose ormonitor the progression of disease, for example, by assaying markerexpression.

For example, an oligonucleotide probe specific for a marker can belabeled by standard methods and added to a biological sample from apatient under conditions that allow for the formation of hybridizationcomplexes. After an incubation period, the sample can be washed and theamount of label (or signal) associated with hybridization complexes canbe quantified and compared with a standard value. If complex formationin the patient sample is significantly altered (higher or lower) incomparison to a normal (e.g., healthy) standard, or is similar to adisease standard, this differential expression can be diagnostic of adisorder. By analyzing changes in patterns of marker expression, diseasemay be diagnosed at earlier stages before a patient is symptomatic. Inexemplary embodiments of the invention, arrays or marker expressionanalysis methods can be used to formulate a diagnosis or prognosis, todesign a treatment regimen, and/or to monitor the efficacy of treatment.For example, a treatment dosage can be established that causes a changein marker expression patterns indicative of successful treatment, andmarker expression patterns associated with the onset of undesirable sideeffects can be avoided. In further exemplary embodiments, assays ofmarker expression can be repeated on a regular basis to determine if thelevel of marker expression in a patient begins to approximate that whichis observed in a normal individual. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to years, for example.

Exemplary arrays of the invention can also be used to screen candidateagents, such as to identify agents that produce a marker expressionprofile similar to that caused by known therapeutic agents, with theexpectation that agents that cause a similar expression profile of amarker may have similar therapeutic effects and/or modes of action onthe marker.

Examples

Exemplary embodiments of the invention are further described in thefollowing examples, which do not limit the scope of the invention.

1. Tissue Samples and Cell Lines

Tissue Processing and Preparation of Single Cell Suspensions from Tissue

Tissue samples (e.g., normal tissues or disease tissues such assurgically resected neoplastic or metastatic lesions) can be procuredfrom clinical sites and transported in transport buffer. Tissues can becollected as remnant tissues following surgical resection of cancer (orother disease) tissues. Remnant tissues are supplied followingprocessing for pathological diagnosis according to proper standards ofpatient care. Normal tissue specimens can be normal tissue adjacent totumors (or other disease tissue) that is collected during tumorresection. Normal tissue from healthy patients not having cancer (orother disease of interest) can also be included, such as to reduce thecontribution from pre-neoplastic changes that may exist in normaladjacent tissue. Procurement of tissue samples is carried out in ananonymous manner in compliance with federally mandated ethical and legalguidelines (HIPAA) and in accordance with clinical institution ethicalreview board and internal institutional review board guidelines.

Tissue can be crudely minced and incubated for 20-30 minutes withperiodic agitation at 37° C. in Enzyme Combination #1 (200 unitscollagenase, cat #C5894 Sigma; 126 μg DNAse I, cat #D4513 Sigma (in 10mM Tris/HCl pH7.5); 50 mM NaCl; 10 mM MgCl2; 0.05% elastase, cat #E7885Sigma) (additionally, hyaluronidase enzyme may also be utilized). D-PBSis added at 3× the volume of the enzyme combination, the tissue finelyminced, and disassociated cells passed through a 200 μm filter. Thecells are washed twice with D-PBS. Red blood cells are lysed withPharMLyse (BD Biosciences) when necessary. Cell number and viability aredetermined by PI exclusion (GUAVA). Cells at a total cell number greaterthan 20×10⁶ are sorted using a high-speed sorter (MoFlo Cytomation) forepithelial cells (EpCAM positive).

The remaining undigested tissue is incubated for 20-30 minutes withperiodic agitation at 37° C. in Enzyme Combination #2 (1× LiberaseBlendzyme 1, cat #988-417 Roche; 1× Liberase Blendzyme 3, cat #814-184Roche; 0.05% elastase, cat #E7885 Sigma). D-PBS is added at 3× thevolume of the enzyme combination, and the tissue finely minced untiltissue is completely disassociated. The cells are passed through a 200μm filter, washed twice with D-PBS, and pooled with cells from theEnzyme Combination #1 digestion. Cells are passed through a 70 μm filterfor single cell suspension, and cell number and viability are determinedby PI exclusion (GUAVA). When needed, red blood cells are lysed withPharMLyse (BD Biosciences). Cells are incubated in 20 ml of 1× PharMLysein D-PBS for 30 seconds with gentle agitation and cells pelleted at300×g for 5 minutes at 4° C. Cells are washed once in D-PBS and cellnumber and viability are recalculated by PI exclusion using the GUAVA.Cells at a total cell number greater than 20×10⁶ are sorted using ahigh-speed sorter (MoFlo Cytomation) for epithelial cells (EpCAMpositive).

Single cell suspensions can also be prepared from tissue samples asfollows: specimens are washed in DTT for 15 min, digested with Dispase(30-60 min), then filtered twice (380 μm/74 μm) before red blood cellsare removed through addition of ACK lysis buffer. Epithelial (EpCAM) andleukocyte (CD45) content and cellular viability (PI exclusion) can bedetermined through flow cytometry analysis (LSR I, BD Biosciences, SanJose, Calif.).

The epithelial content of both disease and normal specimens can beenriched through depletion of immune CD45-positive cells by flowcytometry or purification of Epithelial Cell Surface Antigen(ECSA/EpCam)-positive cells by bead capture.

Bead capture of epithelial cells can be performed using a DynalCELLection Epithelial Enrich kit (Invitrogen, Carlsbad, Calif.) asfollows. Dynal CELLection beads at a concentration of 2×10⁸ beads areincubated with 1×10⁸ cells in HBSS with 10% fetal calf serum for 30minutes at 4° C. Cells and beads are placed in a magnet system Dynal MPCfor 2 minutes. Bead/cell complexes are washed in RPMI 1640 media with 1%fetal calf serum. Cells are released from the bead complex with 15minute incubation with DNase with agitation in RPMI with 1% fetal calfserum.

DynalBead cell depletion of CD45 cells can be carried out as follows.DynalBead M-450 CD45 beads and cells are incubated at a concentration of250 μl beads per 2×10⁷ cells for 30 minutes at 4° C. Bead/cell complexesare washed in DPBS buffer with 2% fetal bovine serum. Cells and beadsare placed in a magnet system Dynal MPC for 2 minutes. The supernatantcontains EpCAM enriched cells.

Cell Line Culture

Cell lines can be obtained from the American Type Culture Collection(ATCC, Manassas, Va.). For example, lung cancer cell lines and normalcontrol lung cell lines (e.g., Beas2B cells can be used as a normalcontrol lung cell line) can be used, such as to determine the expressionlevels of markers (e.g., proteins or encoding mRNA transcripts) in lungcancer cells compared with normal lung cells. Cell lines can be grown ina culturing medium that is supplemented as necessary with growth factorsand serum, in accordance with the ATCC guidelines for each particularcell line. Cultures are established from frozen stocks in which thecells are suspended in a freezing medium (cell culture medium with 10%DMSO [v/v]) and flash frozen in liquid nitrogen. Frozen stocks preparedin this way are stored in liquid nitrogen vapor. Cell cultures areestablished by rapidly thawing frozen stocks at 37° C. Thawed stockcultures are slowly transferred to a culture vessel containing a largevolume of supplemented culture medium. For maintenance of culture, cellsare seeded at 1×10⁵ cells/per ml in medium and incubated at 37° C. untilconfluence of cells in the culture vessel exceeds 50% by area. At thistime, cells are harvested from the culture vessel using enzymes or EDTAwhere necessary. The density of harvested, viable cells is estimated byhemocytometry and the culture reseeded as above. A passage of thisnature is repeated no more than 25 times, at which point the culture isdestroyed and reestablished from frozen stocks as described above.

Alternatively, for secreted protein analysis, cells can be grown underroutine tissue culture conditions in 490 cm² roller bottles at aninitial seeding density of approximately 15 million cells per rollerbottle. When the cells reach ˜70-80% confluence, the culturing media isremoved, the cells are washed 3 times with D-PBS and once with CD293protein-free media (Invitrogen cat #11913-019), and the culturing mediais replaced with CD293 for generating conditioned media. Cells areincubated for 72 hours in CD293 and the media is collected for analysis,such as mass spectrometry analysis of secreted proteins (30-300 ml).Cell debris is removed from the conditioned media by centrifugation at300 g for 5 minutes and filtering through a 0.2 micron filter prior toanalysis.

2. Cloning and Expression of Marker Proteins

cDNA Retrieval

Peptide sequences can be searched using the BLAST algorithm againstrelevant protein sequence databases to identify the correspondingfull-length protein (reference sequence). Each full-length proteinsequence can then be searched using the BLAST algorithm against a humancDNA clone collection. For each sequence of interest, clones can bepulled and streaked onto LB/Ampicillin (100 μg/ml) plates. Plasmid DNAis isolated using Qiagen spin mini-prep kit and verified by restrictiondigest. Subsequently, the isolated plasmid DNA is sequence verifiedagainst the reference full-length protein sequence. Sequencing reactionsare carried out using Applied Biosystems BigDye Terminator kit followedby ethanol precipitation. Sequence data is collected using the AppliedBiosystems 3700 Genetic Analyzer and analyzed by alignment to thereference full-length protein sequence using the Clone Manager alignmenttool.

PCR

PCR primers are designed to amplify the region encoding the full-lengthprotein and/or any regions of the protein that are of interest forexpression (e.g., antigenic or hydrophilic regions as determined by theClone Manager sequence analysis tool). Primers also contain 5′ and 3′overhangs to facilitate cloning (see below). PCR reactions contain 2.5units Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 50 ng cDNAplasmid template, 1 μM forward and reverse primers, 800 μM dNTP cocktail(Applied Biosystems), and 2 mM MgSO₄. After 20-30 cycles (94° C. for 30seconds, 55° C. for 1 minute, and 73° C. for 2 minutes), the resultingproduct is verified by sequence analysis and quantitated by agarose gelelectrophoresis.

Construction of Entry Clones

PCR products are cloned into an entry vector for use with the Gatewayrecombination based cloning system (Invitrogen). These vectors includepDonr221, pDonr201, pEntr/D-TOPO, or pEntr/SD/D-TOPO and are used asdescribed in the cloning methods below.

TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO

For cloning using this method, the forward PCR primer contains a 5′overhang containing the sequence “CACC”. PCR products are generated asdescribed above and cloned into the entry vector using the InvitrogenTOPO® cloning kit. Reactions are typically carried out at roomtemperature for 10 minutes and subsequently transformed into TOP10chemically competent cells (Invitrogen, CA). Candidate clones arepicked, and plasmid DNA is prepared using a Qiagen spin mini-prep kitand screened by restriction enzyme digestion. Inserts are subsequentlysequence-verified as described above.

Gateway Cloning into pDonr201 or pDonr221

For cloning using this method, PCR primers contain forward and reverse5′ overhangs. PCR products are generated as described above.Protein-encoding nucleic acid molecules are recombined into the entryvector using the Invitrogen Gateway BP Clonase enzyme mix. Reactions aretypically carried out at 25° C. for 1 hour, treated with Proteinase K at37° C. for 10 minutes, and transformed into Library Efficiency DH5achemically competent cells (Invitrogen, CA). Candidate clones arepicked, plasmid DNA is prepared using a Qiagen spin mini-prep kit, andscreened by restriction enzyme digestion. Inserts are subsequentlysequence-verified as described above.

Construction of Expression Clones

Protein-encoding nucleic acid molecules are transferred from the entryconstruct into a series of expression vectors using the Gateway LRClonase enzyme mix. Reactions are typically carried out for 1 hour at25° C., treated with Proteinase K at 37° C. for 10 minutes, andsubsequently transformed into Library Efficiency DH5a chemicallycompetent cells (Invitrogen). Candidate clones are picked, plasmid DNAis prepared using a Qiagen spin mini-prep kit, and screened byrestriction enzyme digestion. Expression vectors include, but are notlimited to, pDest14, pDest15, pDest17, pDest8, pDest10 and pDest20.These vectors allow expression in systems such as E. coli andrecombinant baculovirus. Other vectors not listed here allow expressionin yeast, mammalian cells, or in vitro.

Expression of Recombinant Proteins in E. coli

Constructs are transformed into one or more of the following hoststrains: BL21 SI, BL21 AI, (Invitrogen), Origami B (DE3), Origami B(DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Rosetta-Gami (DE3),Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B (DE3) pLysS (Novagen). Thetransformants are grown in LB with or without NaCl and with appropriateantibiotics, at temperatures in the range of 20−37° C., with aeration.Expression is induced with the addition of IPTG (0.03-0.30 mM) or NaCl(75-300 mM) when the cells are in mid-log growth. Growth is continuedfor one to 24 hours post-induction. Cells are harvested bycentrifugation in a Sorvall RC-3C centrifuge in a H6000A rotor for 10minutes at 3000 rpm at 4° C. Cell pellets are stored at −80° C.

Expression of Recombinant Proteins Using Baculovirus

Recombinant proteins are expressed using baculovirus in Sf21 fall armyworm ovarian cells. Recombinant baculoviruses are prepared using theBac-to-Bac system (Invitrogen) per the manufacturer's instructions.Proteins are expressed on the large scale in Sf900II serum-free medium(Invitrogen) in a 10 L bioreactor tank (27° C., 130 rpm, 50% dissolvedoxygen for 48 hours).

3. Recombinant Protein Purification

Recombinant proteins can be purified from E. coli and/or insect cellsusing a variety of standard chromatography methods. Briefly, cells arelysed using sonication or detergents. The insoluble material is pelletedby centrifugation at 10,000×g for 15 minutes. The supernatant is appliedto an appropriate affinity column. For example, His-tagged proteins areseparated using a pre-packed chelating sepharose column (Pharmacia) orGST-tagged proteins are separated using a glutathione sepharose column(Pharmacia). After using the affinity column, proteins are furtherseparated using various techniques, such as ion exchange chromatography(columns from Pharmacia) to separate on the basis of electrical chargeor size exclusion chromatography (columns from Tosohaas) to separate onthe basis of molecular weight, size, and shape.

Expression and purification of the protein can also be achieved usingeither a mammalian cell expression system or an insect cell expressionsystem. The pUB6/V5-His vector system (Invitrogen, CA) can be used toexpress cDNA in CHO cells. The vector contains the selectable bsd gene,multiple cloning sites, the promoter/enhancer sequence from the humanubiquitin C gene, a C-terminal V5 epitope for antibody detection withanti-V5 antibodies, and a C-terminal polyhistidine (6×His) sequence forrapid purification on PROBOND resin (Invitrogen, CA). Transformed cellsare selected on media containing blasticidin.

Spodoptera frugiperda (Sf9) insect cells are infected with recombinantAutographica californica nuclear polyhedrosis virus (baculovirus). Thepolyhedrin gene is replaced with the cDNA by homologous recombinationand the polyhedrin promoter drives cDNA transcription. The protein issynthesized as a fusion protein with 6×His which enables purification asdescribed above. Purified proteins can be used to produce antibodies.

4. Chemical Synthesis of Proteins

Proteins or portions thereof can be produced not only by recombinantmethods (such as described above), but also by using chemical methodswell known in the art. Solid phase peptide synthesis can be carried outin a batchwise or continuous flow process which sequentially addsα-amino- and side chain-protected amino acid residues to an insolublepolymeric support via a linker group. A linker group such asmethylamine-derivatized polyethylene glycol is attached topoly(styrene-co-divinylbenzene) to form the support resin. The aminoacid residues are N-a-protected by acid labile Boc (t-butyloxycarbonyl)or base-labile Fmoc (9-fluorenylmethoxycarbonyl) groups. The carboxylgroup of the protected amino acid is coupled to the amine of the linkergroup to anchor the residue to the solid phase support resin.Trifluoroacetic acid or piperidine are used to remove the protectinggroup in the case of Boc or Fmoc, respectively. Each additional aminoacid is added to the anchored residue using a coupling agent orpre-activated amino acid derivative, and the resin is washed. Thefull-length peptide is synthesized by sequential deprotection, couplingof derivitized amino acids, and washing with dichloromethane and/orN,N-dimethylformamide. The peptide is cleaved between the peptidecarboxy terminus and the linker group to yield a peptide acid or amide.(Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San DiegoCalif. pp. S1-S20).

Automated synthesis can also be carried out on machines such as the 431Apeptide synthesizer (Applied Bio systems, Foster City, Calif.). Aprotein or portion thereof can be purified by preparative highperformance liquid chromatography and its composition confirmed by aminoacid analysis or by sequencing (Creighton, 1984, Proteins, Structuresand Molecular Properties, W H Freeman, New York N.Y.).

5. Antibody Production

Polyclonal Antibodies

Polyclonal antibodies against recombinant proteins can be raised inrabbits (Green Mountain Antibodies, Burlington, Vt.). Briefly, two NewZealand rabbits are immunized with 0.1 mg of antigen in completeFreund's adjuvant. Subsequent immunizations are carried out using 0.05mg of antigen in incomplete Freund's adjuvant at days 14, 21, and 49.Bleeds are collected and screened for recognition of the antigen bysolid phase ELISA and Western blot analysis. The IgG fraction isseparated by centrifugation at 20,000×g for 20 minutes followed by a 50%ammonium sulfate cut. The pelleted protein is resuspended in 5 mM Trisand separated by ion exchange chromatography. Fractions are pooled basedon IgG content. Antigen-specific antibody is affinity purified usingPierce AminoLink resin coupled to the appropriate antigen.

Isolation of Antibody Fragments Directed Against a Marker Protein from aLibrary of scFvs

Naturally occurring V-genes isolated from human PBLs can be constructedinto a library of antibody fragments which contain reactivities againsta marker protein to which the donor may or may not have been exposed(see, for example, U.S. Pat. No. 5,885,793, incorporated herein byreference in its entirety).

Rescue of the library: A library of scFvs is constructed from the RNA ofhuman PBLs, as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 10⁹ E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to innoculate 50 ml of2×TY-AMP-GLU, 2×10⁸ TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 rpm. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 μg/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

Preparation of M13 delta gene III: M13 delta gene III helper phage doesnot encode gene III protein, hence the phage(mid) displaying antibodyfragments have a greater avidity of binding to antigen. Infectious M13delta gene III particles are made by growing the helper phage in cellsharboring a pUC19 derivative supplying the wild type gene III proteinduring phage morphogenesis. The culture is incubated for 1 hour at 37°C. without shaking and then for a further hour at 37° C. with shaking.Cells are spun down (IEC-Centra 8,400 rpm for 10 min), resuspended in300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml(2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particlesare purified and concentrated from the culture medium by twoPEG-precipitations (Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.), resuspended in 2 ml PBS and passed through a 0.45μm filter (Minisart NML; Sartorius) to give a final concentration ofapproximately 10¹³ transducing units/ml (ampicillin-resistant clones).

Panning of the library: Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a marker protein ofinterest. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. andthen washed 3 times in PBS. Approximately 10¹³ TU of phage is applied tothe tube and incubated for 30 minutes at room temperature tumbling on anover-and-under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under-and-over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phagesare then used to infect 10 ml of mid-log E. coli TG1 by incubatingeluted phage with bacteria for 30 minutes at 37° C. The E. coli are thenplated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is then repeated for a total of 4 rounds ofaffinity purification with tube-washing increased to 20 times with PBS,0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of binders: Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks et al., 1991, J. Mol. Biol. 222: 581-597) from singlecolonies for assay. ELISAs are performed with microtitre plates coatedwith either 10 μg/ml of the marker protein of interest in 50 mMbicarbonate pH 9.6. Clones positive in ELISA are further characterizedby PCR fingerprinting (see, e.g., PCT publication WO 92/01047) and thenby sequence analysis.

Monoclonal Antibodies

a) Materials:

1. Complete Media No Sera (CMNS) for washing of the myeloma and spleencells; Hybridoma medium CM-HAT (Cell Mab (BD), 10% FBS (or HS); 5%Origen HCF (hybridoma cloning factor) containing 4 mM L-glutamine andantibiotics) to be used for plating hybridomas after the fusion.

2. Hybridoma medium CM-HT (no aminopterin) (Cell Mab (BD), 10% FBS 5%Origen HCF containing 4 mM L-glutamine and antibiotics) to be used forfusion maintenance is stored in the refrigerator at 4-6° C. The fusionsare fed on days 4, 8, and 12, and subsequent passages. Inactivated andpre-filtered commercial fetal bovine serum (FBS) or horse serum (HS) arethawed and stored in the refrigerator at 4° C. and is pretested formyeloma growth from single cells prior to use.

3. The L-glutamine (200 mM, 100× solution), which is stored at −20° C.,is thawed and warmed until completely in solution. The L-glutamine isdispensed into media to supplement growth. L-glutamine is added to 2 mMfor myelomas and 4 mM for hybridoma media. Further, the penicillin,streptomycin, amphotericin (antibacterial-antifungal stored at −20° C.)is thawed and added to Cell Mab Media to 1%.

4. Myeloma growth media is Cell Mab Media (Cell Mab Media, QuantumYield, from BD, which is stored in the refrigerator at 4° C. in thedark), to which is added L-glutamine to 2 mM and antibiotic/antimycoticsolution to 1% and is called CMNS.

5. One bottle of PEG 1500 in Hepes (Roche, N.J.) is prepared.

6. 8-Azaguanine is stored as the dried powder supplied by SIGMA at −700°C. until needed. One vial/500 ml of media is reconstituted and theentire contents are added to 500 ml media (e.g., 2 vials/liter).

7. Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1×) storedin the refrigerator at 4° C.

8. Clonal cell medium D (Stemcell, Vancouver) contains HAT and methylcellulose for semi-solid direct cloning from the fusion. This comes in90 ml bottles with a CoA and is melted at 37° C. in a waterbath in themorning of the day of the fusion. The cap is loosened and the bottle isleft in a CO₂ incubator to sufficiently gas the medium D and bring thepH down.

9. Hybridoma supplements HT [hypoxanthine, thymidine] to be used inmedium for the section of hybridomas and maintenance of hybridomasthrough the cloning stages, respectively.

10. Origen HCF can be obtained directly from Igen and is a cellsupernatant produced from a macrophage-like cell-line. It can be thawedand aliquoted to 15 ml tubes at 5 ml per tube and stored frozen at −20°C. Positive hybridomas are fed HCF through the first subcloning and aregradually weaned (individual hybridomas can continue to be supplemented,as needed). This and other additives are typically more effective inpromoting new hybridoma growth than conventional feeder layers.

b) Procedure:

To generate monoclonal antibodies, mice are immunized with 5-50 μg ofantigen, either intra-peritoneally (i.p.) or by intravenous injection inthe tail vein (i.v.). The antigen used can be a recombinant markerprotein of interest, for example. The primary immunization takes placetwo months prior to the harvesting of splenocytes from the mouse, andthe immunization is typically boosted by i.v. injection of 5-50 μg ofantigen every two weeks. At least one week prior to the expected fusiondate, a fresh vial of myeloma cells is thawed and cultured. Severalflasks of different densities can be maintained so that a culture at theoptimum density is ensured at the time of fusion. An optimum density canbe 3-6×10⁵ cells/ml, for example. 2-5 days before the scheduled fusion,a final immunization of approximately 5 μg of antigen in PBS isadministered (either i.p. or i.v).

Myeloma cells are washed with 30 ml serum free media by centrifugationat 500 g at 4° C. for 5 minutes. Viable cell density is determined inresuspended cells using hemocytometry and vital stains. Cellsresuspended in complete growth medium are stored at 37° C. during thepreparation of splenocytes. Meanwhile, to test aminopterin sensitivity,1×10⁶ myeloma cells are transferred to a 15 ml conical tube andcentrifuged at 500 g at 4° C. for 5 minutes. The resulting pellet isresuspended in 15 ml of HAT media and cells plated at 2 drops/well on a96-well plate.

To prepare splenocytes from immunized mice, the animals are euthanisedand submerged in 70% ethanol. Under sterile conditions, the spleen issurgically removed and placed in 10 ml of RPMI medium supplemented with20% fetal calf serum in a petri dish. Cells are extricated from thespleen by infusing the organ with medium >50 times using a 21 g syringe.

Cells are harvested and washed by centrifugation (at 500 g at 4° C. for5 minutes) with 30 ml of medium. Cells are resuspended in 10 ml ofmedium and the density of viable cells determined by hemocytometry usingvital stains. The splenocytes are mixed with myeloma cells at a ratio of5:1 (spleen cells: myeloma cells). Both the myeloma and spleen cells arewashed twice more with 30 ml of RPMI-CMNS, and the cells are spun at 800rpm for 12 minutes.

Supernatant is removed and cells are resuspended in 5 ml of RPMI-CMNSand are pooled to fill volume to 30 ml and spun down as before. Then,the pellet is broken up by gently tapping on the flow hood surface andresuspending in 1 ml of BMB REG1500 (prewarmed to 37° C.) dropwise witha 1 cc needle over 1 minute.

RPMI-CMNS to the PEG cells and RPMI-CMNS are added to slowly dilute outthe PEG. Cells are centrifuged and diluted in 5 ml of Complete media and95 ml of Clonacell Medium D (HAT) media (with 5 ml of HCF). The cellsare plated out 10 ml per small petri plate.

Myeloma/HAT control is prepared as follows: dilute about 1000 P3X63Ag8.653 myeloma cells into 1 ml of medium D and transfer into a singlewell of a 24-well plate. Plates are placed in an incubator, with twoplates inside of a large petri plate, with an additional petri platefull of distilled water, for 10-18 days under 5% CO₂ overlay at 37° C.Clones are picked from semisolid agarose into 96-well plates containing150-200 μl of CM-HT. Supernatants are screened 4 days later in ELISA,and positive clones are moved up to 24-well plates. Heavy growthrequires changing of the media at day 8 (+/−150 ml). The HCF can befurther decreased to 0.5% (gradually—2%, then 1%, then 0.5%) in thecloning plates.

6. Liquid Chromatography and Mass Spectrometry (LC/MS)

For LC/MS analysis, proteins are reduced in 2.5 mM DTT for 1 hour at 37°C., and alkylated with ICAT™ reagent according to the proceduresrecommended by the manufacturer (Applied Biosystems, Framingham, Mass.).The reaction is quenched by adding excess DTT. Proteins are digestedusing sequencing grade modified trypsin overnight at 37° C. followed bydesalting using 3 cc Oasis HLB solid phase extraction columns (Waters,Milford, Mass.) and vacuum drying. Cysteine-containing peptides arepurified by avidin column (Applied Biosystems, Framingham, Mass.). Thepeptides are reconstituted in buffer A (0.1% formic acid in water) andseparated over a C18 monomeric column (150 mm, 150 μm i.d., Grace Vydac238EV5, 5 μm) at a flow rate of 1.5 μl/min with a trap column. Peptidesare eluted from the column using a gradient, 3%-30% buffer B (0.1%formic acid in 90% acetonitrile) in 215 min, 30%-90% buffer B in 30 min.Eluted peptides are analyzed using an online QSTAR XL system (MDS/Sciex,Toronto, ON). Peptide ion peaks from the map are automatically detectedwith RESPECT™ (PPL Inc., UK).

The sequence-composition of peptides detected, for example, at higherlevels in disease samples (or drug-resistant samples) relative toadjacent normal tissue (or drug-sensitive samples) can be resolvedthrough tandem mass spectrometry and database analysis. For dataanalysis, peptide ion peaks of LC/MS maps from normal and diseasesamples can be aligned based on mass to charge ratio (m/z), retentiontime (Rt), and charge state (z). The list of aligned peptide ions isloaded into Spotfire™ (Spotfire Inc. Somerville, Mass.). Intensities canbe normalized before further differential analysis between disease andnormal samples. Differentially expressed ions are manually verifiedbefore LC-MS/MS-based peptide sequencing and database searching forprotein/protein identification.

For intensity normalization and expression analysis, a heat map can beconstructed by sorting the rows by the ratio of the mean intensity inthe disease samples to the mean intensity of the normal samples. Rowsare included if there is at least one MS/MS identification of an ion inthe row. The display colors are determined for each row separately byassigning black to the median intensity in the row, green to the lowestintensity in the row, and red to the highest intensity.

Using a mass spectrometry procedure such as this, a comprehensiveanalysis of proteins differentially expressed by disease cells (or drugresistant cells, for example) compared with normal cells (or cellsresponsive/sensitive to a drug, for example) can be carried out.

7. mRNA Expression Analysis

Expression of marker mRNA can be quantitated by RT-PCR using TaqMan®technology. The Taqman® system couples a 5′ fluorogenic nuclease assaywith PCR for real-time quantitation. A probe is used to monitor theformation of the amplification product.

Total RNA can be isolated from disease model cell lines using an RNEasyKit® (Qiagen, Valencia, Calif.) with DNase treatment (per themanufacturer's instructions). Normal human tissue RNAs can be acquiredfrom commercial vendors (e.g., Ambion, Austin, Tex.; Stratagene, LaJolla, Calif.; BioChain Institute, Newington, N.H.), as well as RNAsfrom matched disease/normal tissues.

Marker transcript sequences can be identified for differentiallyexpressed peptides by database searching using a search algorithm suchas BLAST. TaqMan® assays (PCR primer/probe sets) specific for thosetranscripts can be obtained from Applied Biosystems (AB) as part of theAssays on Demand™ product line or by custom design through the AB Assaysby Design℠ service. If desired, the assays can be designed to spanexon-exon borders so as not to amplify genomic DNA.

RT-PCR can be accomplished using AmpliTaq Gold® and MultiScribe™ reversetranscriptase in the One Step RT-PCR Master Mix reagent kit (AB)(according to the manufacturer's instructions). Probe and primerconcentrations are 250 nM and 900 nM, respectively, in a 15 μl reaction.For each experiment, a master mix of the above components is made andaliquoted into each optical reaction well. Eight nanograms of total RNAis used as template. Quantitative RT-PCR can be performed using the ABIPrism® 7900HT Sequence Detection System (SDS). The following cyclingparameters are used: 48° C. for 30 min. for one cycle; 95° C. for 10 minfor one cycle; and 95° C. for 15 sec, 60° C. for 1 min. for 40 cycles.

SDS software can be utilized to calculate the threshold cycle (C_(T))for each reaction, and C_(T) values are used to quantitate the relativeamount of starting template in the reaction. The C_(T) values for eachset of reactions can be averaged for all subsequent calculations

Data can be analyzed to determine estimated copy number per cell. Geneexpression can be quantitated relative to 18S rRNA expression and copynumber estimated assuming 5×10⁶ copies of 18S rRNA per cell.Alternatively, data can be analyzed for fold difference in expressionusing an endogenous control for normalization and expressed relative toa normal tissue or normal cell line reference. The choice of endogenouscontrol can be determined empirically by testing various candidatesagainst the cell line and tissue RNA panels and selecting the one withthe least variation in expression. Relative changes in expression can bequantitated using the 2^(−ΔΔCT) method (Livak et al., 2001, Methods 25:402-408; User bulletin #2: ABI Prism 7700 Sequence Detection System).Alternatively, total RNA can be quantitated using a RiboGreen RNAQuantitation Kit according to manufacturer's instructions and thepercentage mRNA expression calculated using total RNA for normalization.Percentage knockdown can then be calculated relative to a no additioncontrol.

8. Flow Cytometry (FACS) Analysis

Flow cytometry is interchangeably referred to as fluorescence-activatedcell sorting (FACS). Quantitative flow cytometry can be used to comparethe level of expression of a protein on disease cells to the level foundon normal cells, for example.

Expression levels of a marker protein on primary tissue samples can bequantified using the Quantum Simply Cellular System (Bangs Laboratories,Fishers, Ind.) and a marker-specific antibody. Normal adjacent anddisease tissues can be processed into single cell suspensions, asdescribed above, which can be stained for various markers (e.g., theepithelial marker EpCam) and the marker-specific antibody. At least0.5×10⁶ cells are typically used for each analysis. Cells are washedonce with Flow Staining Buffer (0.5% BSA, 0.05% NaN3 in D-PBS). To thecells, 20 μl of each marker-specific antibody are added. An additional 5μl of anti-EpCam antibody conjugated to APC can be added when unsortedcells are used. Cells are incubated with antibodies for 30 minutes at 4°C. Cells are washed once with Flow Staining Buffer and either analyzedimmediately on an LSR flow cytometry apparatus or fixed in 1%formaldehyde and stored at 4° C. until LSR analysis. Antibodies used todetect a marker can be PE-conjugated. PE-conjugated mouse IgG1k can usedas an isotype control antibody. Cells are analyzed by flow cytometry andepitope copy number and the percentage of viable epithelial cellspositive for marker expression can be measured. Cell numbers andviability can be determined by PI exclusion (GUAVA) for cells isolatedfrom both normal and disease tissue. Standard curve and samples can beanalyzed on a LSR I (BDBiosciences, San Jose Calif.) flow cytometer.Antibody binding capacity for each lineage population can be calculatedusing geometric means and linear regression.

Expression levels of a marker protein can be quantified in cell lineswith QIFIKIT flow cytometric indirect immunofluorescence assay (DakoA/S) using a primary antibody to the marker. Briefly, cells are detachedwith versene or trypsin and washed once with complete media and thenPBS. 5×10⁵ cells/sample are incubated with saturating concentration (10μg/ml) of primary antibody for 60 minutes at 4° C. After washes, aFITC-conjugated secondary antibody (1:50 dilution) is added for 45minutes at 4° C. QIFIKIT standard beads are simultaneously labeled withthe secondary antibody. Binding of antibodies is analyzed by flowcytometry and specific antigen density is calculated by subtractingbackground antibody equivalent from antibody-binding capacity based on astandard curve of log mean fluorescence intensity versus log antigenbinding capacity.

Cells can also be prepared for flow cytometry analysis (as well as othertypes of analysis) as follows: cells are incubated with 1:100 dilutionof BrdU in culturing media for 2-4 hours (BrdU Flow Kit, cat #559619 BDBiosciences). Cells are washed 3 times with D-PBS and disassociated fromthe flask with versene. Cell numbers and viability can be determined byPI exclusion (GUAVA). Cells are washed once with Flow Staining Buffer(0.5% BSA, 0.05% NaN3 in D-PBS). Cells are incubated with 400 μl ofCytofix/Cytoperm Buffer (BrdU Flow Kit, BD Biosciences) for 15-30minutes at 4° C. Cells are washed once with Flow Staining Buffer andresuspended in 400 μl Cytoperm Plus Buffer (BrdU Flow Kit BDBiosciences). Cells are incubated for 10 minutes at 4° C. and washedonce with 1×Perm/Wash Buffer (BrdU Flow Kit, BD Biosciences). Cells areincubated for 1 hour at 37° C. protected from light in DNAse solution(BrdU Flow Kit, BD Biosciences). Cells are washed once with 1× Perm/WashBuffer and incubated for 20 min at room temperature with anti-BrdUFITC-conjugated antibody (BrdU Flow Kit, BD Biosciences), PE-conjugatedactive caspase 3 (BD Biosciences cat #550821), and PE mouse IgG2Bisotype control. Cells are washed once with 1×Perm/Wash Buffer andresuspended in DAPI for LSR flow cytometry analysis.

9. Immunohistochemistry (IHC)

IHC of Tissue Sections

Paraffin embedded, fixed tissue sections (e.g., from disease tissuesamples such as solid tumors or other cancer tissues) can be obtainedfrom a panel of normal tissues as well as tumor (or other disease)samples with matched normal adjacent tissues, along with replicatesections (if desired). For example, for an initial survey of markerexpression, a panel of common cancer formalin-fixed paraffin-embedded(FFPE) tissue microarrays (TMAs) can be used for analysis, and such TMAscan be obtained from commercial sources (TriStar, Rockville, Md.;USBiomax, Rockville, Md.; Imgenex, San Diego, Calif.; Petagen/Abxis,Seoul, Korea). Sections can be stained with hemotoxylin and eosin andhistologically examined to ensure adequate representation of cell typesin each tissue section. An identical set of tissues can be obtained fromfrozen sections for use in those instances where it is not possible togenerate antibodies that are suitable for fixed sections. Frozen tissuesdo not require an antigen retrieval step.

Paraffin Fixed Tissue Sections

An exemplary protocol for hemotoxylin and eosin staining of paraffinembedded, fixed tissue sections is as follows. Sections aredeparaffinized in three changes of xylene or xylene substitute for 2-5minutes each. Sections are rinsed in two changes of absolute alcohol for1-2 minutes each, in 95% alcohol for 1 minute, followed by 80% alcoholfor 1 minute. Slides are washed in running water and stained in Gillsolution 3 hemotoxylin for 3-5 minutes. Following a vigorous wash inrunning water for 1 minute, sections are stained in Scott's solution for2 minutes. Sections are washed for 1 minute in running water and thencounterstained in eosin solution for 2-3 minutes, depending upon thedesired staining intensity. Following a brief wash in 95% alcohol,sections are dehydrated in three changes of absolute alcohol for 1minute each and three changes of xylene or xylene substitute for 1-2minutes each. Slides are coverslipped and stored for analysis.

Optimization of Antibody Staining

For each antibody, a positive and negative control sample can begenerated using data from ICAT analysis of disease cell lines ortissues. Cells can be selected that are known to express low levels of aparticular marker as determined from the ICAT data, and this cell linecan be used as a reference normal control. Similarly, a disease cellline that is determined to over-express the marker can also be selected.

Antigen Retrieval

Sections are deparaffinized and rehydrated by washing 3 times for 5minutes in xylene, two times for 5 minutes in 100% ethanol, two timesfor 5 minutes in 95% ethanol, and once for 5 minutes in 80% ethanol.Sections are then placed in endogenous blocking solution (methanol+2%hydrogen peroxide) and incubated for 20 minutes at room temperature.Sections are rinsed twice for 5 minutes each in deionized water andtwice for 5 minutes in phosphate buffered saline (PBS), pH 7.4.

Alternatively, where necessary, sections are de-parrafinized by HighEnergy Antigen Retrieval as follows: sections are washed three times for5 minutes in xylene, two times for 5 minutes in 100% ethanol, two timesfor 5 minutes in 95% ethanol, and once for 5 minutes in 80% ethanol.Sections are placed in a Coplin jar with dilute antigen retrievalsolution (10 mM citrate acid, pH 6). The Coplin jar containing slides isplaced in a vessel filled with water and microwaved on high for 2-3minutes (700 watt oven). Following cooling for 2-3 minutes, steps 3 and4 are repeated four times (depending on the tissue), followed by coolingfor 20 minutes at room temperature. Sections are then rinsed indeionized water (two times for 5 minutes), placed in modified endogenousoxidation blocking solution (PBS+2% hydrogen peroxide), and rinsed for 5minutes in PBS.

Alternatively, formalin fixed paraffin embedded tissues can bedeparaffinized and processed for antigen retrieval using theEZ-retriever system (BioGenex, San Ramon, Calif.). EZ-antigen Retrievalcommon solution is used for deparaffinization and EZ-retrievalcitrate-based buffer used for antigen retrieval. Samples are pre-blockedwith non-serum protein block (Dako A/S, Glostrup, Denmark) for 15 min.Primary antibodies (at 2.5-5.0 μg/ml, for example) are incubatedovernight at room temperature. Envision Plus system HRP (Dako A/S) isused for detection with diaminobenzidine (DAB) as substrate forhorseradish peroxidase.

Blocking and Staining

Sections are blocked with PBS/1% bovine serum albumin (PBA) for 1 hourat room temperature followed by incubation in normal serum diluted inPBA (2%) for 30 minutes at room temperature to reduce non-specificbinding of antibody. Incubations are performed in a sealed humiditychamber to prevent air-drying of the tissue sections. The choice ofblocking serum is typically the same as the species of the biotinylatedsecondary antibody. Excess antibody is gently removed by shaking andsections covered with primary antibody diluted in PBA and incubatedeither at room temperature for 1 hour or overnight at 4° C. (care istaken that the sections do not touch during incubation). Sections arerinsed twice for 5 minutes in PBS, shaking gently. Excess PBS is removedby gently shaking. The sections are covered with diluted biotinylatedsecondary antibody in PBA and incubated for 30 minutes to 1 hour at roomtemperature in the humidity chamber. If using a monoclonal primaryantibody, addition of 2% rat serum can be used to decrease thebackground on rat tissue sections. Following incubation, sections arerinsed twice for 5 minutes in PBS, shaking gently. Excess PBS is removedand sections incubated for 1 hour at room temperature in Vectastain ABCreagent (as per kit instructions). The lid of the humidity chamber issecured during all incubations to ensure a moist environment. Sectionsare rinsed twice for 5 minutes in PBS, shaking gently.

Developing and Counterstaining

Sections are incubated for 2 minutes in peroxidase substrate solutionthat is made up immediately prior to use as follows: 10 mgdiaminobenzidine (DAB) dissolved in 10 ml of 50 mM sodium phosphatebuffer, pH 7.4; 12.5 microliters 3% CoCl₂/NiCl₂ in deionized water; and1.25 microliters hydrogen peroxide.

Slides are rinsed well three times for 10 minutes in deionized water andcounterstained with 0.01% Light Green acidified with 0.01% acetic acidfor 1-2 minutes, depending on the desired intensity of counterstain.

Slides are rinsed three times for 5 minutes with deionized water anddehydrated two times for 2 minutes in 95% ethanol; two times for 2minutes in 100% ethanol; and two times for 2 minutes in xylene. Stainedslides are mounted for visualization by microscopy.

Slides are scored manually using a microscope such as the Zeiss Axiovert200M microscope (Carl Zeiss Microimaging, Thornwood, N.Y.).Representative images are acquired using 40× objective (400×magnification).

IHC Staining of Frozen Tissue Sections

For IHC staining of frozen tissue sections, fresh tissues are embeddedin OCT in plastic mold, without trapping air bubbles surrounding thetissue. Tissues are frozen by setting the mold on top of liquid nitrogenuntil 70-80% of the block turns white at which point the mold is placedon dry ice. The frozen blocks are stored at −80° C. Blocks are sectionedwith a cryostat with care taken to avoid warming to greater than −10° C.Initially, the block is equilibrated in the cryostat for about 5 minutesand 6-10 mm sections are cut sequentially. Sections are allowed to dryfor at least 30 minutes at room temperature. Following drying, tissuesare stored at 4° C. for short term and −80° C. for long term storage.

Sections are fixed by immersing in an acetone jar for 1-2 minutes atroom temperature, followed by drying at room temperature. Primaryantibody is added (diluted in 0.05 M Tris-saline [0.05 M Tris, 0.15 MNaCl, pH 7.4], 2.5% serum) directly to the sections by covering thesection dropwise to cover the tissue entirely. Binding is carried out byincubation in a chamber for 1 hour at room temperature. Without lettingthe sections dry out, the secondary antibody (diluted inTris-saline/2.5% serum) is added in a similar manner to the primaryantibody and incubated as before (at least 45 minutes).

Following incubation, the sections are washed gently in Tris-saline for3-5 minutes and then in Tris-saline/2.5% serum for another 3-5 minutes.If a biotinylated primary antibody is used, in place of the secondaryantibody incubation, slides are covered with 100 μl of diluted alkalinephosphatase conjugated streptavidin, incubated for 30 minutes at roomtemperature and washed as above. Sections are incubated with alkalinephosphatase substrate (1 mg/ml Fast Violet; 0.2 mg/ml Napthol AS-MXphosphate in Tris-Saline pH 8.5) for 10-20 minutes until the desiredpositive staining is achieved at which point the reaction is stopped bywashing twice with Tris-saline. Slides are counter-stained with Mayer'shematoxylin for 30 seconds and washed with tap water for 2-5 minutes.Sections are mounted with Mount coverslips and mounting media.

10. RNAi Assays in Cell Lines

RNAi Transfections

Expression of a marker can be knocked down by transfection with smallinterfering RNA (siRNA) to that marker. Synthetic siRNA oligonucleotidescan be obtained from Dharmacon (Lafayette, Colo.) or Qiagen (Valencia,Calif.). For siRNA transfection, cells (e.g., disease cells) can beseeded into 96 well tissue culture plates at a density of 2,500 cellsper well 24 hours before transfection. Culture medium is removed and 50μl of reaction mix containing siRNA (final concentration 1 to 100 nM)and 0.4 μl of DharmaFECT4 (Dharmacon, Lafayette, Colo.) diluted inOpti-MEM is added to each well. An equal volume of complete mediumfollows and the cells are then incubated at 5% CO₂ at 37° C. for 1 to 4days.

Alternatively, in the initial screening phase, RNAi can be performedusing 100 nM (final) of Smartpools (Dharmacon, Lafayette, Colo.), poolof 4—for Silencing siRNA duplexes (Qiagen, Valencia, Calif.), ornon-targeting negative control siRNA (Dharmacon or Qiagen). In thebreakout phase, each individual duplex is used at 100 nM (final). In thetitration phase, individual duplex is used at 0.1-100 nM (final).Transient transfections are carried out using either Lipofectamine 2000from Invitrogen (Carlsbad, Calif.) or GeneSilencer from Gene TherapySystems (San Diego, Calif.) (see below). One day after transfections,total RNA is isolated using the RNeasy 96 Kit (Qiagen) according tomanufacturer's instructions and expression of mRNA is quantitated usingTaqMan technology. Apoptosis and cell proliferation assays can beperformed daily using Apop-one homogeneous caspase-3/7 kit and AlamarBlue or CellTiter 96 AQueous One Solution Cell Proliferation Assays (seebelow).

RNAi Transfections—Lipofectamine 2000 and GeneSilencer

Transient RNAi transfections can be carried out using Lipofectamine 2000(Invitrogen, Carlsbad, Calif.) or GeneSilencer (Gene Therapy Systems,San Diego, Calif.), such as on sub-confluent disease cell lines, asdescribed elsewhere (Elbashir et al., 2001, Nature 411: 494-498; Caplenet al., 2001, Proc Natl Acad Sci USA 98: 9742-9747; Sharp, 2001, Genesand Development 15: 485-490). Synthetic RNA to a gene of interest ornon-targeting negative control siRNA are transfected using Lipofectamine2000 or GeneSilencer according to manufacturer's instructions. Cells areplated in 96-well plates in antibiotic-free medium. The next day, thetransfection reagent and siRNA are prepared for transfections asfollows.

0.1-100 nM siRNA is resuspended in 20-25 μl serum-free media in eachwell (with Plus for Lipofectamine 2000) and incubated at roomtemperature for 15 minutes. 0.1-1 μl of Lipofectamine 2000 or 1-1.5 μlof GeneSilencer is also resuspended in serum-free medium to a finalvolume of 20-25 μl per well. After incubation, the diluted siRNA andeither the Lipofectamine 2000 or the GeneSilencer are combined andincubated for 15 minutes (Lipofectamine 2000) or 5-20 minutes(GeneSilencer) at room temperature. Media is then removed from the cellsand the combined siRNA-Lipofectamine 2000 reagent or siRNA-GeneSilencerreagent is added to a final volume of 50 μl per well. After furtherincubation at 37° C. for 4 hours, 50 μl serum-containing medium is addedback to the cells. 1-4 days after transfection, expression of mRNA canbe quantitated by RT-PCR using TaqMan technology, and protein expressionlevels can be measured by flow cytometry. Apoptosis and proliferationassays can be performed daily using Apop-one homogeneous caspase-3/7 kitand Alamar Blue or CellTiter 96 AQueous One Solution Cell ProliferationAssays (see below).

mRNA and Protein Knockdowns

Knockdown of marker mRNA levels can be monitored by Q-PCR one day aftersiRNA transfection by using a TaqMan® assay (Applied Biosystems, FosterCity, Calif.). RT-PCR is accomplished in a one-step reaction by usingM-MLV reverse transcriptase (Promega, Madison, Wis.) and AmpliTaq Gold®(ABI) and analyzed on the ABI Prism® 7900HT Sequence Detection System(ABI). Relative gene expression can be quantitated by the ΔΔCt method(User Bulletin #2, ABI) with 18S rRNA serving as the endogenous control.

Protein knockdown can be monitored by FACS four days after transfectionby using an antibody to the marker. The samples can be run on a LSR flowcytometer (BD Biosciences, San Jose, Calif.) and live cells monitored byusing PI exclusion (50 μg/ml PI, 2.5 units/ml RNase A, 0.1% Triton X-100in D-PBS). The data can be analyzed using CellQuest software.

Cell Proliferation—Alamar Blue

Cell growth can be assessed four days after transfection by adding a1:10 dilution of Alamar blue reagent (Invitrogen, Carlsbad, Calif. orBiosource, Camarillo, Calif.) and incubated for 2 hours at 37° C.Analysis can be performed on a Spectrafluor Plus (Tecan, Durham, N.C.)set at excitation wavelength of 530 nm and emission wavelength of 595nm.

Cell Proliferation—MTS

Alternatively, cell proliferation assays can be performed using aCellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega,Madison, Wis.). 20 μl of CellTiter 96 AQueous One Solution is added to1000 of culture medium. The plates are then incubated for 1-4 hours at37° C. in a humidified 5% CO₂ incubator. After incubation, the change inabsorbance is read at 490 nm.

Apoptosis

Apoptosis assays can be performed using the Apop-one homogeneouscaspase-3/7 kit (Promega, Madison, Wis.). Briefly, the caspase-3/7substrate is thawed to room temperature and diluted 1:100 with buffer.The diluted substrate is then added 1:1 to cells, control, or blank. Theplates are then placed on a plate shaker for 30 minutes to 18 hours at300-500 rpm. The fluorescence of each well is then measured using anexcitation wavelength of 485+/−20 nm and an emission wavelength of530+/−25 nm.

11. Antibody Assays in Cell Lines

Cytotoxicity Assays

Cytotoxicity can be measured using a Resazurin (Sigma, Mo.) dyereduction assay (McMillian et al., 2002, Cell Biol. Toxicol.18:157-173). Briefly, cells are plated at 1,000-5,500 cells/well in 96well plates, allowed to attach to the plates for 18 hours beforeaddition of fresh media with or without antibody. After 96-144 hours ofexposure to antibody, resazurin is added to cells to a finalconcentration of 50 Cells are incubated for 2-6 hours depending on dyeconversion of cell lines, and dye reduction is measured on a Fusion HTfluorescent plate reader (Packard Instruments, Meridien, Conn.) withexcitation and emission wavelengths of 530 nm and 590 nm, respectively.The IC₅₀ value is defined here as the drug concentration that results in50% reduction in growth or viability as compared with untreated controlcultures.

Assays for Antibody-Dependent Cellular Cytotoxicity

Antibody-dependent cellular cytotoxicity (ADCC) assays can be carriedout as follows. Cultured disease cells (e.g., tumor cells) are labeledwith 100 μCi ⁵¹Cr for 1 hour (Livingston et al., 1997, Cancer Immunol.Immunother. 43, 324-330). After being washed three times with culturemedium, cells are resuspended at 10⁵/ml, and 100 μl/well are plated onto96-well round-bottom plates. A range of antibody concentrations areapplied to the wells, including an isotype control together with donorperipheral blood mononuclear cells that are plated at a 100:1 and 50:1ratio. After an 18 hour incubation at 37° C., supernatant (30 μl/well)is harvested and transferred onto Lumaplate 96 (Packard), dried, andread in a Packard Top-Count NXT γ counter. Spontaneous release isdetermined by cpm of disease cells incubated with medium and maximumrelease by cpm of disease cells plus 1% Triton X-100 (Sigma). Specificlysis is defined as: % specific lysis=[(experimental release−spontaneousrelease)/(maximum release−spontaneous release)]×100. The percent ADCC isexpressed as peak specific lysis postimmune subtracted by preimmunepercent specific lysis. A doubling of the ADCC to >20% can typically beconsidered significant.

Assays for Complement Dependent Cytotoxicity

Chromium release assays to assess complement dependent cytotoxicity(CDC) can be carried out as follows (Dickler et al., 1999, Clin. CancerRes. 5, 2773-2779). Cultured disease cells (e.g., tumor cells) arewashed in FCS-free media two times, resuspended in 500 μl of media, andincubated with 100 μCi ⁵¹Cr per 10 million cells for 2 hours at 37° C.The cells are then shaken every 15 min for 2 hours, washed 3 times inmedia to achieve a concentration of approximately 20,000 cells/well, andthen plated in round-bottom plates. The plates contain either 50 μlcells plus 50 μl monoclonal antibody, 50 μl cells plus serum (pre- andpost-therapy), or 50 μl cells plus mouse serum as a control. The platesare incubated in a cold room on a shaker for 45 min. Human complement ofa 1:5 dilution (resuspended in 1 ml of ice-cold water and diluted with3% human serum albumin) is added to each well at a volume of 100 Controlwells include those for maximum release of isotope in 10% Triton X-100(Sigma) and for spontaneous release in the absence of complement withmedium alone. The plates are incubated for 2 hours at 37° C.,centrifuged for 3 min, and then 100 μl of supernatant is removed forradioactivity counting. The percentage of specific lysis is calculatedas follows: % cytotoxicity=[(experimental release-spontaneousrelease)/(maximum release-spontaneous release)]×100. A doubling of theCDC to >20% can typically be considered significant.

Cell Proliferation Assays

To measure cell proliferation, cells can be plated, grown and treated asfor the cytotoxicity assay (above) in 96 well plates. After 96-144 hoursof treatment, 0.5 μCi/well ³H-Thymidine (PerkinElmer, 6.7 Ci/mmol) isadded to cells and incubated for 4-6 hours at 37° C., 5% CO₂ in anincubator. To lyse cells, plates are frozen overnight at −20° C. andthen cell lysates are harvested using FilterMate (Packard Instrument,Meridien, Conn.) into 96 well filter plates. Radioactivity associatedwith cells is measured on a TopCount (Packard) scintillation counter.

Other cell assays (e.g., proliferation assays such as Alamar blue andMTS, and apoptosis assays) can be carried out using antibodies, asdescribed above for RNAi.

Testing of Function-Blocking Antibodies

For testing of function-blocking antibodies, sub-confluent disease celllines are serum-starved overnight. The next day, serum-containing mediais added back to the cells in the presence of 5-50 ng/ml offunction-blocking antibodies. After 2 or 5 days incubation at 37° C. 5%CO₂, antibody binding is examined by flow cytometry, and apoptosis andproliferation are measured.

Cell Invasion

Cell invasion assays can be performed using a 96-well cell invasionassay kit (Chemicon). After the cell invasion chamber plates areadjusted to room temperature, 100 μl serum-free media is added to theinterior of the inserts. 1-2 hours later, cell suspensions of 1×10⁶cells/ml are prepared. Media is then carefully removed from the insertsand 100 μl of prepared cells are added into the insert+/−0 to 50 ngfunction blocking antibodies. The cells are pre-incubated for 15 minutesat 37° C. before 150 μl of media containing 10% FBS is added to thelower chamber. The cells are then incubated for 48 hours at 37° C. Afterincubation, the cells from the top side of the insert are discarded andthe invasion chamber plates are then placed on a new 96-well feeder traycontaining 150 μl of pre-warmed cell detachment solution in the wells.The plates are incubated for 30 minutes at 37° C. and are periodicallyshaken. Lysis buffer/dye solution (4 μl CyQuant Dye/300 μl 4× lysisbuffer) is prepared and added to each well of dissociation buffer/cellson feeder tray. The plates are incubated for 15 minutes at roomtemperature before 150 μl is transferred to a new 96-well plate.Fluorescence of invading cells is then read at 480 nm excitation and 520nm emission.

Receptor Internalization

For quantification of receptor internalization, ELISA assays can beperformed essentially as described by Daunt et al. (Daunt et al., 1997,Mol. Pharmacol. 51, 711-720). Cell lines are plated at 6×10⁵ cells perin a 24-well tissue culture dishes that have previously been coated with0.1 mg/ml poly-L-lysine. The next day, the cells are washed once withPBS and incubated in DMEM at 37° C. for several minutes. Agonist to thecell surface marker of interest is then added to the wells at apre-determined concentration in prewarmed DMEM. The cells are thenincubated for various times at 37° C. and reactions are stopped byremoving the media and fixing the cells in 3.7% formaldehyde/TBS for 5min at room temperature. The cells are then washed three times with TBSand nonspecific binding blocked with TBS containing 1% BSA for 45 min atroom temperature. The first antibody is added at a pre-determineddilution in TBS/BSA for 1 hr at room temperature. Three washes with TBSfollow, and cells are briefly reblocked for 15 min at room temperature.Incubation with goat anti-mouse conjugated alkaline phosphatase(Bio-Rad) diluted 1:1000 in TBS/BSA is carried out for 1 hr at roomtemperature. The cells are washed three times with TBS and acolorimetric alkaline phosphatase substrate is added. When the adequatecolor change is reached, 100 μl samples are taken for colorimetricreadings.

12. Treatment with Antibodies

Treatment of Disease Cells with Monoclonal Antibodies.

Disease cells (e.g., cancer cells), or cells such as NIH 3T3 cells thatexpress a marker of interest, are seeded at a density of 4×10⁴ cells perwell in 96-well microtiter plates and allowed to adhere for 2 hours. Thecells are then treated with different concentrations of monoclonalantibody (Mab) specific for the marker protein of interest, orirrelevant isotype matched (e.g., anti-rHuIFN-gamma) Mab, at 0.05, 0.5or 5.0 μg/ml. After a 72 hour incubation, the cell monolayers arestained with crystal violet dye for determination of relative percentviability (RPV) compared to control (untreated) cells. Each treatmentgroup can have replicates. Cell growth inhibition is monitored.

In Vivo Treatment with Monoclonal Antibodies.

NIH 3T3 cells transfected with either an expression plasmid thatexpresses the marker of interest or a neo-DHFR vector are injected intonu/nu (athymic) mice subcutaneously at a dose of 10⁶ cells in 0.1 ml ofphosphate-buffered saline. On days 0, 1, 5, and every 4 days thereafter,100 μg (0.1 ml in PBS) of a Mab specific for the marker protein ofinterest, or an irrelevant Mab, of the IgA2 subclass is injectedintraperitoneally. Disease progression (e.g., tumor occurrence and size)can be monitored for a one month period of treatment, for example.

13. Identification of LCM

A mass spectrometry (MS)-based proteomics platform was used for theidentification of secreted and shed proteins (secreted and shed proteinsare collectively referred to herein as soluble proteins) and cellsurface antigens that combines the discovery of candidate biomarkersfrom human lung tumor specimens resected from surgery and in a panel oflung cancer cell lines, followed by validation of expression levels inpatient serum (such as by using ELISA). For example, proteomic analysistechniques such as MALDI-TOF/TOF LC/MS-based protein expression analysiswas used to determine the expression levels of certain proteins in lungtumor tissues and/or lung cancer cell lines (tissues and cell lines maybe collectively referred to herein as “samples”) and in normal tissuesand/or normal cell lines, such that proteins that are differentiallyexpressed (e.g., over- or under-expressed) in lung cancer samplescompared with normal samples were identified.

Certain candidate markers were identified by mass spectrometry-basedmethods that were differentially expressed on the cell surface of lungtumors, lung cancer cell lines, or secreted into the conditioned mediumof cell lines. Certain of these candidate markers that were identifiedas differentially expressed by mass spectrometry, as well as certainother candidate markers, were assayed by ELISA and scored in panels oflung cancer patient sera and sera from individuals without lung cancer(individuals without lung cancer are referred to herein as “normal”,“control”, or “healthy” individuals). Individual markers were scored“positive” for a given cancer sample if the value exceeded a definedthreshold (e.g., greater than or equal to two standard deviations abovethe mean value for a group of “normal” samples tested). From thesecandidate markers, lung cancer markers (“LCM”) were identified that,particularly when used in combination, distinguished lung cancer samplesfrom healthy control samples with various degrees of sensitivity andspecificity.

Several methods and algorithms were applied to select optimumpanels/combinations of LCM including sum of the logs of the ratios ofthe tumor concentration to the mean of the normal concentration,defining a concentration cutoff manually for each marker to optimizesensitivity and specificity, and use of Naïve Bayes to assign aprobability that a sample is a tumor based on the expression level ofeach marker. Additionally, ROC curves may be constructed for each paneland their effectiveness may be evaluated in several ways, such asmaximizing the AUC of the ROC curve as well as maximizing thesensitivity at a desired specificity or maximizing the specificity at adesired sensitivity.

To further validate the specificity of certain panels, co-morbiditystudies were carried out to challenge certain panels with other lungdisease samples besides lung cancer, particularly chronic obstructivepulmonary disease (COPD), asthma, bronchitis, and other benign lungdiseases (FIG. 20). Prevalence of COPD/asthma is 10-25% in smokers. Aninitial panel of 30 bronchitis/asthma/benign lung disease samples wastested. Results indicated that these co-morbidities may reducespecificity only marginally if considered independent of false positivesin 54 control samples (the specificity of the 9-member panel of Cyfra,SLPI, TIMP1, SCC, TFPI, CEACAM5, MMP2, OPN, and MDK in a 54 normal/53lung tumor sample set was 98% on samples from smoking controls).

14. ELISA Immunoassays

Immunoassay kits, such as for performing ELISA assays, for various LCMdisclosed herein are commercially available. For example, immunoassaykits can be obtained from a variety of commercial sources, as follows:SLPI, MMP2, MIF, and OPN immunoassay kits can be obtained from R&DSystems (Minneapolis, Minn.); CYFRA 21-1 and SCC immunoassay kits can beobtained from DRG-International (Mountainside, N.J.); DEFA1 immunoassaykits can be obtained from Cell Sciences (Canton, Mass.); TIMP1immunoassay kits can be obtained from Siemens Healthcare Diagnostics(Cambridge, Mass.); CEA and GRP immunoassay kits can be obtained fromIBL International (Toronto, Ontario); TFPI immunoassay kits can beobtained from American Diagnostica (Stamford, Conn.); and MDKimmunoassay kits can be obtained from BioVendor (Candler, N.C.) or R&DSystems (Minneapolis, Minn.). Assays can be performed followingmanufacturers instructions. Plates can be read on a Spectra Max M2Microplate Reader (Molecular Devices, Sunnyvale, Calif.) with theappropriate baseline correction for each assay.

HNP1-3 (defensin, DEFA1) is employed as a representative marker in thefollowing exemplary ELISA protocol, which can be used for the analysisof LCM. An HNP1-3 ELISA test kit can be used that is a solid-phaseenzyme-linked immunosorbent assay based on the sandwich principle.Samples and standards are incubated in microtiter wells coated withantibodies recognizing human HNP1-3. During this incubation, humanHNP1-3 is captured by solid bound antibody. Unbound material present inthe sample is removed by washing. Biotinylated second antibody (tracer)to human HNP 1-3 is then added to the wells. If HNP1-3 is present in thesample, the tracer antibodies will bind to the captured HNP1-3. Theexcess tracer is removed by washing. A streptavidin-peroxidase conjugateis then applied to the wells, which reacts specifically with thebiotinylated tracer antibody bound onto the detected HNP1-3. The excessstreptavidin-peroxidase conjugate is removed by washing and substratetetramethylbenzidine (TMB) is added to the wells. Color developsproportionally to the amount of human HNP1-3 present in the sample. Theenzyme reaction is stopped by the addition of citric acid and theabsorption at 450 nm is measured with a spectrophotometer. A standardcurve is obtained by plotting the absorptions versus the correspondingconcentrations of the known standards. The concentration of human HNP1-3in test samples, which are run concurrently with the standards, can bedetermined from the standard curve.

15. Scoring of LCM Levels

This example describes an exemplary method of scoring LCM levels usingsplit-point analysis.

The term “split-point analysis” refers to a method adapted from Mor etal., PNAS, (2005) 102, 7677-7682. In this exemplary method, measurementsfor each marker are taken on all samples. A cutoff value is determinedfor each marker. This cutoff value may be set to, for example, maximizethe accuracy of correct classifications between the groups of interest(e.g., tumor and control sample groups) or may be set to maximize thesensitivity or specificity of one group. For each marker, a score isassigned to that sample whenever the value of that marker is found to beon the diseased side of the cutoff value (e.g., the side of the cutoffcorresponding to lung tumor samples). After all the measurements havebeen taken on one sample, the scores are summed to produce a total scorefor the panel of markers. All markers can be weighted equally such thata panel of 9 markers may have a maximum score of 9 (each marker having ascore of either 1 or 0) and a minimum score of 0, for example.Alternatively, markers can be weighted unequally, with a higherindividual score for more significant measures.

Other more sophisticated statistical modeling methods can also beapplied such as logistic regression (see, e.g., Planque et al., ClinCancer Res (2008), 14, 1355-1362) and decision tree modeling (see, e.g.,Patz et al., J Clin Oncol (2007), 25, 5578-5583).

An exemplary method of applying split-point analysis to an LCM panel isdescribed for illustrative purposes.

A patients sample can be tested to determine the patient's likelihood ofhaving lung cancer using a panel comprising the 9 biomarkers Cyfra, CEA,SLPI, OPN, MDK, TFPI, TIMP1, MMP2, and SCC and the split and scoremethod. The predetermined total score (or threshold) for the panel canbe set at 1 (or other value).

After obtaining a test sample from the patient, the amount of each ofthe 9 biomarkers (Cyfra, CEA, SLPI, OPN, MDK, TFPI, TIMP1, MMP2, SCC) inthe patient's test sample is quantified. For the purpose of thisexample, the amount of each of the 9 biomarkers in the test sample isdetermined to be as follows (values are expressed in ng/ml):Cyfra=0.891, CEA=4.087, SLPI=62.94, OPN=21.514, MDK=0.174, TFPI=104.503,TIMP1=398.7, MMP2=194.41, and SCC=1.35. The amount of each of thesebiomarkers is then compared to the corresponding predetermined cutoff(or split point). For the purpose of this example, the predeterminedcutoffs for each of the biomarkers are as follows: Cyfra=1.20, CEA=5.00,SLPI=52, OPN=32, MDK=0.15, TFPI=150, TIMP1=385, MMP2=210, and SCC=2.2.For each biomarker having an amount that is higher than itscorresponding predetermined cutoff (split point), a score of 1 can beassigned. For each biomarker having an amount that is less than or equalto its corresponding predetermined cutoff, a score of 0 can be assigned.Thereupon, based on said comparison, each biomarker would be assigned ascore as follows: Cyfra=0, CEA=0, SLPI=1, OPN=0, MDK=1, TFPI=0, TIMP1=1,MMP2=0, and SCC=0.

The score for each of the 9 biomarkers can then be combinedmathematically (e.g., by adding each of the scores of the biomarkerstogether) to arrive at the total score for the patient. In thisparticular example, the total score for the patient is 3 (the totalscore is calculated as follows: 0+0+1+0+1+0+1+0+0=3). The total scorefor the patient is compared to the predetermined total score, which is 1in this particular example. A total score greater than the predeterminedtotal score of 1 would indicate a positive result for the patient (i.e.,in this particular example, a total score of 2 or greater would indicatethat the patient has lung cancer). A total score equal to or less than 1would indicate a negative result for the patient. In this example,because the patient's total score is greater than 1, the patient wouldbe considered to have a positive result (and thus may be referred forfurther testing for an indication or suspicion of lung cancer). Incontrast, had the patient's total score been 1 or 0, the patient wouldhave been considered to have a negative result (and thus would not bereferred for any further testing).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described methods and compositions of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific exemplary embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention, which are obviousto those skilled in the field of molecular biology or related fields,are intended to be within the scope of the following claims.

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LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US11105806B2).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

That which is claimed is:
 1. A method comprising detecting the levels oftissue factor pathway inhibitor (TFPI) protein, carcinoembryonicantigen-related cell adhesion molecule 5 (CEACAM5) protein, and Cyfra21-1 (Cyfra) protein in a body fluid sample from a human, detecting anelevated lung cancer biomarker panel when the level of at least one ofsaid proteins is elevated, and administering a therapeutic agent totreat lung cancer to said human when said elevated lung cancer biomarkerpanel is detected.
 2. The method of claim 1, further comprisingdetecting the level of squamous cell carcinoma antigen (SCC).
 3. Themethod of claim 1, wherein the method comprises analyzing said levels bysplit-point analysis or logistic regression analysis.
 4. The method ofclaim 3, wherein said split-point analysis or said logistic regressionanalysis is performed by computer software.
 5. The method of claim 1,wherein the level of at least one of said proteins which is elevated isat or above a predetermined cutoff level.
 6. The method of claim 5,wherein said predetermined cutoff level comprises a number of standarddeviations above the normal mean level of said protein established forindividuals who do not have lung cancer.
 7. The method of claim 6,wherein said number of standard deviations is two standard deviations.8. The method of claim 1, wherein the level of each of said proteins isadded together to obtain a total value.
 9. The method of claim 8,wherein the total value is at or above a predetermined cutoff value. 10.The method of claim 8, wherein the level of each of said proteins isanalyzed by split-point analysis or logistic regression analysis toobtain said total value.
 11. The method of claim 1, wherein the level ofat least two of said proteins is elevated.
 12. The method of claim 1,wherein the level of all three of said proteins is elevated.
 13. Themethod of claim 1, wherein the level of said TFPI protein is elevated.14. The method of claim 1, wherein the level of said TFPI protein and atleast one other of said proteins is elevated.
 15. The method of claim 1,wherein the detecting comprises detecting the proteins by immunoassay.16. The method of claim 15, wherein the immunoassay comprises ELISA. 17.The method of claim 1, wherein the detecting comprises contacting thesample with antibodies that selectively bind to each of the proteins anddetecting the binding of the antibodies to the proteins.
 18. The methodof claim 1, wherein the detecting comprises detecting the proteins bymass spectrometry.
 19. The method of claim 1, wherein the detectingcomprises contacting the sample with aptamers that selectively bind toeach of the proteins and detecting the binding of the aptamers to theproteins.
 20. The method of claim 1, wherein said proteins are detectedby reagents which are configured in a multiplex format.
 21. The methodof claim 1, wherein the body fluid sample is blood, serum, plasma, orbronchial lavage.
 22. The method of claim 1, wherein said body fluidsample is serum or plasma, and wherein the method further comprisesseparating said serum or said plasma from a blood sample from saidhuman.
 23. The method of claim 1, wherein said human has been identifiedas having a lung nodule prior to said detecting.
 24. The method of claim23, wherein said lung nodule was detected by computed tomography (CT)screening.
 25. The method of claim 23, further comprising classifyingsaid lung nodule as either malignant or benign.
 26. The method of claim1, further comprising performing computed tomography (CT) screening ofsaid human when said elevated lung cancer biomarker panel is detected.27. The method of claim 1, wherein the method is carried out followingcomputed tomography (CT) screening of said human.
 28. The method ofclaim 1, wherein the method is carried out following radiation therapyor surgical resection of at least a portion of a lung tumor.
 29. Themethod of claim 1, further comprising determining one or moresupplemental biomedical parameters.
 30. The method of claim 29, whereinsaid supplemental biomedical parameters include lung nodule size.
 31. Amethod comprising detecting the levels of proteins consisting of tissuefactor pathway inhibitor (TFPI) protein, carcinoembryonicantigen-related cell adhesion molecule 5 (CEACAM5) protein, and Cyfra21-1 (Cyfra) protein in a body fluid sample from a human, detecting anelevated lung cancer biomarker panel when the level of at least one ofsaid proteins is elevated, and administering a therapeutic agent totreat lung cancer to said human when said elevated lung cancer biomarkerpanel is detected.